Cytotoxicity mediation of cells evidencing surface expression of MCSP

ABSTRACT

This invention relates to the diagnosis and treatment of cancerous diseases, particularly to the mediation of cytotoxicity of tumor cells; and most particularly to the use of cancerous disease modifying antibodies (CDMAB), optionally in combination with one or more chemotherapeutic agents, as a means for initiating the cytotoxic response. The invention further relates to binding assays which utilize the CDMABs of the instant invention.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/949,846, filed Sep. 24, 2004, which is a continuation-in-part of U.S.patent application Ser. No. 10/810,744, filed Mar. 26, 2004, now U.S.Pat. No. 7,393,531, issued Jul. 1, 2008, which is a continuation-in-partof U.S. patent application Ser. No. 10/762,129, filed Jan. 20, 2004, nowU.S. Pat. No. 7,361,342, issued Apr. 22, 2008, which is acontinuation-in-part of U.S. patent application Ser. No. 10/743,451,filed Dec. 19, 2003, now abandoned, which is a continuation of U.S.patent application Ser. No. 10/348,231, filed Jan. 21, 2003, now U.S.Pat. No. 7,009,040, issued Mar. 7, 2006, the contents of each of whichare herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the diagnosis and treatment of cancerousdiseases, particularly to the mediation of cytotoxicity of tumor cells;and most particularly to the use of cancerous disease modifyingantibodies (CDMAB), optionally in combination with one or morechemotherapeutic agents, as a means for initiating the cytotoxicresponse. The invention further relates to binding assays, which utilizethe CDMAB of the instant invention.

BACKGROUND OF THE INVENTION

Melanoma-associated chondroitin sulfate proteoglycan (MCSP) wasidentified independently by several investigators who developedmonoclonal antibodies to human metastatic melanoma cell lines. Severalantibodies were found to react with a specific antigen associated withthe melanoma cell surface. The independent development of theseantibodies led to the multiplicity of names for the target antigen, allof which were subsequently determined to be MCSP. MCSP has thereforealso been referred to as high molecular weight melanoma associatedantigen (HMW-MAA), human melanoma proteoglycan (HMP),melanoma-associated proteoglycan antigen (MPG) and melanoma chondroitinsulfate proteoglycan (mel-CSPG), and has been identified as the antigenof various specific antibodies, some of which have been set out below.MCSP was also found to be over 80 percent homologous with the ratproteoglycan NG2 and is hence also referred to by that name.

MCSP is a glycoprotein-proteoglycan complex consisting of an N-linkedglycoprotein of 250 kDa and a proteoglycan component >450 kDa. The coreglycoprotein is present on the surface of melanoma cells, either as afree glycoprotein or modified by the addition of chondroitin sulfate.The molecular cloning of MCSP led to the identification of severalstructural features. There are 3 extracellular domains containing atotal of 10 cysteines (5 potential disulfide bridges), 15 possibleN-linked glycosylation sites, and 11 potential chondroitin sulfateattachment sites. The transmembrane segment has a single cysteine,however the functional significance of that residue has not beenestablished. The cytoplasmic domain has 3 threonine residues that mayserve as sites for phosphorylation by protein kinase C, although it hasnot yet been shown that MCSP is phosphorylated.

It has been shown that MCSP is expressed in the majority of melanomacancers, and it was originally thought that it had a very limiteddistribution on normal cells and other tumor types. One early study thatled to this conclusion used immunohistochemistry (IHC) on normal andtumor tissues fixed with formaldehyde or methanol in order to determinethe distribution of MCSP using anti-MCSP antibody B5. In this study,antibody B5 was found to react with 17 out of 22 melanoma tumors tested,2 out of 2 astrocytomas tested, and none of the 23 carcinomas tested.Out of 22 normal tissues tested, B5 was found to bind only skinkeratinocytes, lung alveolar epithelium and capillary endothelium.

Another study examined the tissue distribution of MCSP as defined byanti-MCSP antibody 9.2.27 using frozen tissue sections. Again,reactivity was found in all melanoma tissues and cell lines tested, butthere was no reactivity in any of the 6 various carcinoma tumors tested.Out of the 7 fetal tissues tested, reactivity was only observed in theskin and faintly in the aorta while in adult tissues, reactivity wasonly seen in 3 out of 13 tissues tested.

A subsequent study examined the distribution of MCSP using the anti-MCSPantibodies B5, 9.2.27, 225.28S and A0122, all of which recognizedistinct epitopes of MCSP. This study was performed on frozen tissues.It was found that all of the anti-MCSP antibodies had similar stainingpatterns, reacting with normal and malignant tumors of neural,mesenchymal and epithelial origin, that were previously thought to beMCSP negative. Specifically, the antibody B5 reacted with variousepithelial, connective, neural and muscular tissues in the 24 organsthat were tested, and reacted with 28 out of 34 various tumors tested.The authors explained that the differences between their findings andprevious reports were due to the use of improved and more consistent IHCtechniques, noting that choice of fixative was important, presumablyleading to the conclusion that an important characteristic of the MCSPantigen is its sensitivity to the processing steps involved in IHC.

A further study was carried out in order to localize MCSP at theultrastructural level. Immunolocalization studies using electronmicroscopy demonstrated that MCSP was localized almost exclusively tomicrospikes, a microdomain of the melanoma cell surface that may play arole in cell-cell contact and cell-substratum adhesion.

The molecular cloning of MCSP in 1996 enabled northern blot analysis ofMCSP expression in tumor cell lines and normal human tissues using MCSPcDNA probes. Out of 8 various tumor cell lines tested, expression ofMCSP was observed only in the melanoma cell line. MCSP expression wasnot seen in any of the 16 normal adult and 4 normal fetal tissuestested. The discrepancies found in different studies of tissuelocalization of MCSP indicate that further study may be required toelucidate the actual expression patterns of this antigen or to accountfor the differences that have been reported.

Since proteoglycans have been known to mediate cell-cell andcell-extracellular matrix (ECM) interactions, the role of MCSP in theseprocesses has been investigated. MCSP has been shown to stimulateα₄β₁-integrin mediated adhesion and spreading of melanoma cells, and ithas also been proposed that signaling through the MCSP core proteininduces recruitment and tyrosine phosphorylation of p130^(cas) which mayregulate cell adhesion and motility, contributing to tumor invasion andmetastasis. The combination of these results indicated that MCSP mayfunction to enhance adhesion of melanoma cells by both activatingintegrins and stimulating pathways that lead to cytoskeletalrearrangement.

MCSP has also been found to associate with membrane-type 3 matrixmetalloproteinase (MT3-MMP), likely through the chondroitin sulfatecomponent of MCSP. It has been suggested that MT3-MMP expression inmelanomas in vivo could promote the degradation of ECM proteins in thevicinity of the growing tumor, providing space in which the tumor canexpand. Therefore, the association between MT3-MMP and MCSP may be anactivation step to promote melanoma invasion.

Several in vitro assays using anti-MCSP antibodies have been carried outto examine the role of MCSP in processes linked to tumor invasion andmetastasis. The role of MCSP in anchorage-independent growth wasassessed using the antibody 9.2.27. Melanoma cells cultured in soft agarcontaining 9.2.27 showed a 67-74 percent specific decrease in theircolony formation. These findings suggested that MCSP might be involvedin cell-cell interaction, and contribute to anchorage-independentgrowth. The same authors also examined the effects of blocking MCSP with9.2.27 in assays measuring the adhesion of M14 melanoma cells onbasement membranes of bovine aorta endothelial (BAE) cells. The effectof 9.2.27 treatment was compared to treatment with a control monoclonalantibody W6/32 (directed against all class I histocompatibilityantigens). M14 control cells and M14 cells pretreated with antibody wereplated on basement membranes of BAE cells. A significant inhibition of27 percent in cell adhesion was observed in 9.2.27 treated cells,whereas no significant effect was observed in W6/32 treated cells. Amore striking effect of cell pretreatment with 9.2.27 was the inhibitionof cell spreading which was verified at the ultrastructural level usingscanning electron microscopy.

Many of the antibodies that were developed against melanoma cells anddetermined to specifically recognize MCSP have been tested in both invitro and in vivo assays to determine their anti-cancer effects.

Monoclonal antibody 9.2.27 recognizes the core glycoprotein component ofMCSP and was one of the first antibodies investigated for tumorsuppressing properties. Bumol et al. investigated 9.2.27 and adiphtheria toxin A (DTA) conjugate of 9.2.27 for immunotherapy ofmelanoma tumors grown in nude mice. In vitro cytotoxicity assays werefirst carried out by measuring the effects of both 9.2.27 and 9.2.27-DTAconjugate on protein synthesis in M21 human melanoma cells as indicatedby protein incorporation of [³⁵S]methionine. The 9.2.27-DTA conjugatesignificantly inhibited protein synthesis in M21 melanoma cells, thougha greater effect was seen with unconjugated DTA. There was only minimaleffect achieved by 9.2.27 alone. Both the 9.2.27 and 9.2.27-DTAconjugate were investigated for anti-tumor effects in human melanomatumor-bearing nude mice. M21 tumor mince was implanted subcutaneouslyand allowed to establish growth for 3 days, then mice were treated atday 3 and at 3 day intervals thereafter with either 9.2.27 or 9.2.27-DTAconjugate. Tumor volumes of treated mice were compared to those ofuntreated control mice. At day 18 (the last day for which data wasreported), 9.2.27 treated mice showed a 64 percent inhibition of tumorgrowth while 9.2.27-DTA conjugate treated mice showed a 52 percentinhibition of tumor growth, compared to untreated controls. In thisinitial study the authors concluded that 9.2.27 and 9.2.27-DTA conjugatewere approximately equivalent in their effect on suppression of growthof M21 melanoma tumors in nude mice. While this initial study reports invivo suppression of tumor growth by treatment with 9.2.27, severalsubsequent studies, including those by the same authors, havedemonstrated that naked 9.2.27 did not exhibit any anti-tumor effects invivo. Collectively, as outlined below, the experiments carried out toinvestigate the utility of using 9.2.27 to treat human tumors havedemonstrated that, although cancer cells were targeted by 9.2.27, noanti-cancer activity resulted from treatment with the naked antibody.

A phase I clinical trial was carried out which was designed to givelarge doses of 9.2.27 in anticipation of later therapeutic studies with9.2.27 immunoconjugates. Eight patients with malignant melanoma whosetumors reacted with 9.2.27 by flow cytometry and/or immunoperoxidasestaining, received single doses of antibody intravenously, twice weeklyon a dose escalating scale of 1, 10, 50, 100 and 200 mg. Although noneof the patients experienced significant toxicity and 9.2.27 localized tothe metastatic melanoma nodules, no clinical responses were observed.

In a later study, 9.2.27 was conjugated to the chemotherapeutic drugdoxorubicin (DXR), and the conjugate was investigated for growthinhibition of melanoma in vitro and in vivo. Growth inhibition of M21cells treated with the DXR-9.2.27 conjugate was measured using a[³H]thymidine incorporation assay. The conjugate showed specificdose-dependent growth inhibition of the M21 target cells and no effecton an MCSP negative control cell line. No in vitro assays were carriedout examining effects of 9.2.27 alone. To investigate the DXR-9.2.27conjugate in vivo, M21 cells were injected subcutaneously and allowed toestablish a tumor for 8-10 days. Injections were given intravenously atday 10 and at 3 day intervals thereafter for 30 days. Significantsuppression of tumor growth was seen only in mice treated with theDXR-9.2.27 conjugate. Both DXR treatment alone and 9.2.27 treatmentalone failed to suppress tumor growth; a mixture of 9.2.27 and DXRshowed similar negative effects.

Another study was carried out investigating the effects of a 9.2.27conjugate. Schrappe et al. conjugated the chemotherapeutic agent4-desacetylvinblastine-3-carboxyhydrazide (DAVLBHY) to 9.2.27 and testedits effect on human gliomas. Nude mice were injected with U87MG (a humanglioma cell line) cells subcutaneously and the animals were treated ondays 2, 5, 7, and 9. Tumor volume was most effectively reduced by the9.2.27-DAVLBHY conjugate. Control groups, which were treated with eitherPBS or 9.2.27 alone, developed fast growing tumors and there was noreduction in tumor volume in 9.2.27 treated mice compared to micetreated with PBS.

Antibody 225.28S was made against the human M21 melanoma cell line, andwas initially described as reacting with a high molecular weightmelanoma associated antigen. This molecule was subsequently shown to bethe same molecule as MCSP. An early study tested the cytolytic abilityof 225.28S, an IgG_(2a), on a human melanoma cell line and compared itto another anti-MCSP antibody, clone 653.40S that was an IgG₁. 225.28Sand 653.40S were determined to recognize the same, or spatially close,antigenic determinants on MCSP. It was found that neither antibody couldlyse melanoma cells in conjunction with complement in in vitro assays.Both antibodies could mediate lysis of target melanoma cells in anantibody-dependent cell-mediated (ADCC) cytotoxicity assay, with 225.28Sexhibiting a higher lytic activity than 653.40S. However, lysis ofmelanoma cells was only obtained with a significantly highereffector/target cell ratio than had been reported by others usinganti-melanoma antigen antibodies. The authors concluded that the lack ofcytolytic activity of these antibodies in conjunction with humancomplement and the high effector/target cell ratio required for lysis tooccur in ADCC suggested that the injection of monoclonal antibodies intomelanoma patients was not likely to cause the destruction of tumorcells. The authors suggested that the immunotherapeutic use of theseantibodies should be limited to utilizing them as carriers ofradioisotope, chemotherapeutic or toxic agents.

Naked antibody 225.28S was investigated for its therapeutic potential ina phase I trial where it was delivered intravenously in 10 mg doses to 2patients with end-stage melanoma. Although no clinically adverse ormajor toxic effects were noted that could be ascribed to theadministration of the antibody, there was also no positive therapeuticresponse.

Antibody 225.28S was conjugated to purothionin, a low molecular weightpolypeptide that is especially toxic to dividing cells, and was testedfor its in vitro toxicity to the human melanoma cell line Colo 38. Itwas found that the culture of Colo 38 cells with the 225.28S-purothioninconjugate for 24 hr inhibited ³H-thymidine uptake. In addition, theviability of Colo 38 cells was dramatically reduced in culturesincubated with the conjugate for 7 days. Although in vitro toxicity wasobserved, there was still a fraction of melanoma cells that survived the225.28S-purothionin treatment. The authors suggested that theimmunotherapy of malignant diseases may have to rely on cocktails ofmonoclonal antibodies to distinct tumor associated antigens as carriersof toxic agents, indicating that 225.8S conjugate alone would not besufficient for treatment of cancer. The effect of 225.28S-purothioninconjugate treatment was evaluated on the growth of human melanoma innude mice. Colo 38 cells were implanted either subcutaneously orintraperitoneally in nude mice. Treatments were made on days 1, 3 and 5for the intraperitoneal implanted mice and on days 1, 3, 5 and 20 forthe subcutaneous implanted mice. Survival was monitored for all mice.The only statistically significant prolongation of survival was observedin the intraperitoneal implanted mice that were treated with the225.28S-purothionin conjugate. 225.28S alone, purothionin alone or amixture of 225.28S and purothionin did not enhance survival in eitherthe intraperitoneal or the subcutaneous implanted mice. Tumor volume wasalso recorded for the subcutaneous implanted mice and it was found thatonly the 225.28S-purothionin conjugate treatment significantly reducedtumor volume. Treatment with 225.28S alone did not result in a reductionof tumor volume.

225.28S was also conjugated to the chemotherapeutic drug methotrexate(MTX) and its effects on tumor growth were investigated in vivo. Nudemice were inoculated subcutaneously with M21 human melanoma cells andtreated on days 1, 4, 7, 10 and 14. The MTX-225.28S conjugate was theonly treatment that inhibited tumor growth. 225.28S alone, MTX alone ora mixture of 225.28S and MTX failed to inhibit tumor growth.

225.28S was used in a study designed to investigate the potential toxiceffects in humans due to the administration of a reagent of a xenogenicnature. 85 patients with metastatic cutaneous melanoma were administeredeither intact 225.28S or the F(ab′)₂ fragment that were labeled with¹³¹I, ¹²³I, ¹¹¹In, or ⁹⁹Tc. The amount of injected antibody ranged from14 to 750 μg. No clinically detectable side effects were observed in anyof the patients. No clinical response was reported, though it was notnecessarily anticipated as this study was designed for toxologicpurposes.

225.28S was used to generate murine anti-idiotypic monoclonal antibodiesincluding the antibody MF11-30, which bears the mirror image of MCSP.MF11-30 has been shown to induce the development of anti-MCSP antibodiesin both a syngeneic and xenogeneic system. MF11-30 was tested in 2clinical trials in escalating doses designed to test the toxicity andresponse in patients with advanced malignant melanoma. In both studiesthere were few side effects due to administration of the antibody andthe therapy was well tolerated. In the second trial the average survivalof 7 patients who developed anti-anti-idiotypic antibodies with a titerof at least 1:8 and displayed no marked changes in the level of serumMCSP was 55 weeks (range 16-95), which was significantly higher than theremaining 12 patients (who developed anti-antidiotypic antibodies with atiter of 1:4 or less and displayed an increase in the serum level ofMCSP) whose average survival was 19 weeks (range 8-57).

Antibody 763.74 was also generated against melanoma cells and recognizesMCSP. There have not been any reports of in vitro or in vivo anti-cancereffects of antibody 763.74, however this antibody was also used togenerate murine anti-idiotypic monoclonal antibodies. One of theseantibodies, MK2-23, bears the internal image of the determinant definedby the anti-MCSP antibody 763.74. In preclinical experiments,immunization with MK2-23 was shown to induce the development ofanti-MCSP antibodies in both a syngeneic host (BALB/c mice) and in axenogenic host (rabbit). The immunogenicity of MK2-23 was markedlyenhanced when it was conjugated to the carrier protein keyhole limpethemocyanin (KLH) and administered with an adjuvant. A clinical trial wascarried out to characterize the humoral response induced by MK2-23 inpatients with melanoma. 25 patients with stage 1V melanoma wereimmunized on days 0, 7 and 28 with 2 mg subcutaneous injections ofMK2-23 conjugated to KLH and mixed with Bacillus Calmette Guerin (BCG).Additional injections were given if the titer of anti-anti-idiotypicantibodies was low. Approximately 60 percent of the patients who wereimmunized with MK2-23 developed anti-MCSP antibodies, although the leveland affinity of the anti-MCSP antibodies were low. It was found thatsurvival of patients who developed anti-MCSP antibodies afterimmunization with MK2-23 was significantly longer than those who didnot. The median survival of patients who developed anti-MCSP antibodieswas 52 weeks (range 19-93) while the median survival of the 9 patientswithout detectable anti-MCSP antibodies in their sera was 19 weeks(range 9-45). Three patients who developed anti-MCSP antibodiesexperienced a partial remission of their disease. Although promisingresults were achieved in this study, 40 percent of the patientsimmunized with MK2-23 did not respond with detectable anti-MCSPantibodies. As well, the 3 patients who had achieved partial remissionall eventually experienced recurrence of disease. An attempt was made toincrease the immunogenicity of MK2-23 by pretreatment of patients with alow dose of cyclophosphamide (CTX), which had been reported to enhancethe cellular and humoral response to tumor associated antigens byselectively inactivating some sets of suppressor cells. However, noeffects of pretreatment with CTX on the immunogenicity of MK2-23 weredetected.

Monoclonal antibody 48.7 was developed against the human metastaticmelanoma cell line M1733 and was reported to react against a moleculesubsequently determined to be MCSP. 48.7 was administered in a phase Iclinical trial in combination with the murine monoclonal antibody 96.5,which is directed against the transferrin-like cell surface glycoproteinp97 that is present on human melanomas. Five patients received 2 mg eachof mAbs 96.5 and 48.7 on day 1, 10 mg each on day 2, and 25 mg each ondays 3 through 10. Treatment was well tolerated; however there were noclinical responses to treatment and disease progression occurred in allpatients. These two antibodies were investigated in a separate clinicaltrial where 3 patients with melanoma metastatic to the central nervoussystem were treated with radiolabeled Fab fragments of either one ofthese antibodies. Two patients received 5 mg doses of ¹³¹I-labeled Fabfragment of 48.7 in conjunction with osmotic opening of the blood-brainbarrier (BBB) in an effort to enhance entry of the antibody into tumorsin the brain. The osmotic BBB modification increased the delivery of Fabto the tumor-bearing hemisphere and spinal fluid, but clear persistentlocalization of the antibody to the tumor was not shown. The authorshypothesized that the lack of localization may have been due to aninadequate dose of the antibody.

Melimmune was a dual preparation of two murine anti-idiotypicantibodies, Melimmune-1 (1-MeI-1) and Melimmune-2 (1-MeI-2), which mimicseparate epitopes of MCSP. I-MeI-1 was a subclone of the anti-idiotypicantibody MF11-30, which was developed against the anti-MCSP antibody225.28 (as previously discussed). I-MeI-1 was shown to induce ananti-MCSP response in rabbits. I-MeI-2 was an anti-idiotypic antibodydeveloped against the anti-MCSP antibody MEM136, which reacts to adifferent epitope on MCSP than does 225.28. I-MeI-2 was also shown toinduce an anti-MCSP response in rabbits. The Melimmune preparation,which contained a 1:1 composition of 1-MeI-1 and I-MeI-2, was tested ina phase I trial of 21 patients with resected melanoma without evidenceof metastatic disease. Detailed immune response analysis was reportedfor 12 of these patients enrolled in a single institution. Patientsreceived Melimmune on 1 of 2 treatment schedules with doses that rangedfrom 0.2 to 4.0 mg (0.1 to 2.0 mg each of 1-MeI-1 and I-MeI-2). Allpatients developed both anti-1-MeI-1 and anti-1-MeI-2 antibodies withthe peak antibody response to I-MeI-2 greater than that to I-MeI-1 in 10out of 12 patients. However, this study was unable to demonstrateinduction of specific antibodies to MCSP since none of the patient'ssera was able to inhibit either binding of radiolabeled 225.28 to MCSPexpressing MeI-21 cells, or binding of radiolabeled MEM136 to MeI-21cells. A direct cell binding assay was also used to assay for thepresence of anti-MCSP antibodies in patients sera; however, there was nodifference in the binding of preimmune sera compared to postimmune serato M21 cells in a FACS based assay.

I-MeI-2 was tested in a separate clinical trial where 26 patients withmetastatic melanoma were treated with 2 mg I-MeI-2 and either 100 or 250μg of the adjuvant SAF-m delivered intramuscularly biweekly for 4 weeksand then bimonthly until disease progression. Anti-MCSP antibodies weredetected in 5 of the 26 patients using an inhibition radioimmunoassay.Of the 5 patients with detectable anti-MCSP antibodies, 1 patientexperienced a complete remission, 1 had stable disease and the other 3had progressive disease. The patient with complete remission had thehighest titer of anti-MCSP antibodies (1:1500).

Prior Patents:

U.S. Pat. No. 5,270,202 (and its related patents: WO9216646A1,EP0576570A1) teaches an anti-idiotypic antibody, IMelpg2 (also known as“IM32”) to MEM136, an antibody directed to human melanoma-associatedproteoglycan (also known as “HMW-MAA”). The IMelpg2 antibody was shownto be directed to MEM136 specifically, and suggested to be of use forthe diagnosis and treatment of disease in which cells expressed the MPGepitope. Although there was an effect of IMelpg2 on tumor cell invasion,as determined by in vitro assays it was neither the most effectiveantibody tested, nor was there indications of in vivo anti-tumor effectsdespite showing an Ab3 response.

EP0380607B1 teaches anti-idiotypic antibodies to the Mab 225.28 whichhas specificity for an undefined epitope of HMW-MAA. These antibodiesare useful as active immunotherapy for melanoma. Both MF11-30 andIMelpg1, and polyclonal anti-idiotypic antibodies to 225.28 have beenreported and evaluated in animal models with MF11-30 undergoing clinicaltrials in melanoma patients, although there was no supporting data.MF11-30 can induce 225.28 idiotypic antibodies. The IMelpg1 cell linewas derived from treating the MF11-30 cell line with BM Cycline andtesting for the absence of mycoplasma contamination. Although antibodiesto IMelpg1 can be induced in rabbit sera, and be shown to bind to theColo38 melanoma cell, there was no indication of tumorcidal activity,either in vitro or in vivo.

U.S. Pat. No. 4,879,225 teaches the production of antibodies frominsoluble immune complexes. In this case rat anti-idiotypic antibodiesto Mab 9.2.27, an antibody directed against the HMW-MAA, were generatedby immobilizing 9.2.27 on protein A-Sepharose for use as an antigen.Antibodies to melanoma cells were produced using a variety of cell orcell lysate complexes conjugated to Sepharose. Murine monoclonalantibodies that bound to melanoma cells, but not normal T-cells orB-cells were compared to 9.2.27. Those that had similar properties to9.2.27 were selected for further characterization: NR-ML-02, NR-ML-03,NR-ML-04, NR-ML-05, NR-ML-06. Each of these antibodies were positive ina sandwich ELISA assay using 9.2.27 as the capture antibody andsolublized SK MEL-28 melanoma membranes as an antigen source. Furtherthese antibodies were characterized as recognizing melanoma tumor cells,and also reacting with smooth muscle and endothelial cells. Anadditional 61 anti-proteoglycan antibodies were produced with 10recognizing the same determinant as NR-ML-02/NR-ML-04, 3 antibodiesrecognized the same determinant as NR-ML-03 or NR-ML-05; 45 did notrecognize the same epitope as determined by the 5 antibodies. In U.S.Pat. No. 5,084,396 these antibodies were radiolabelled and compared with9.2.27 for tumor uptake in nude mice bearing melanoma xenografts. Thetumor uptake was the greatest for NR-ML-05 and NR-ML-02, then 9.2.27,and then NR-ML-02. In neither of these inventions were there indicationsthat these antibodies produced reduction in tumor burden of cancerousdisease, nor enhanced survival of mammals having cancerous disease.

U.S. Pat. No. 5,034,223 teaches a method of enhancing delivery ofconjugated antibodies to tissues bearing tumor-associated antigens bypretreating with a non-conjugated blocking antibody. Antibodies toHMW-MAA, 9.2.27 and NR-ML-05, were conjugated to technicium 99 (Tc-99)and were administered in the clinical setting after prior administrationof unlabelled Mab NR-2AD, an antibody with an anti-idiotype specific foronly 1 patient's B-cell lymphoma. Since these studies were designedusing Tc-99 as a reporter radioisotope, which does not have cytotoxic,or radioablative effects there was no evidence of anti-tumor effectsalthough there was enhanced uptake of the anti-HMW-MAA antibodiesthrough the use of this process.

U.S. Pat. No. 5,580,774 teaches the construction of a chimeric antibodyusing the antibody genes that encode Mab 9.2.27. No disclosuresregarding the diagnosis or treatment of cancerous disease using thechimeric antibody were made.

U.S. Pat. No. 5,493,009 and U.S. Pat. No. 5,780,029 teaches the murineanti-idiotypic antibody MK2-23, and its conjugates, directed against ananti-HMW-MAA antibody, 763.74. MK2-23 can bind directly to 763.74 andinhibit 763.74 binding to Colo 38 melanoma cells. Further, Ab3 elicitedby MK2-23 can directly bind HMW-MAA and can competitively inhibit 763.74binding to Colo 38 melanoma cells. Active immunotherapy was carried outin a clinical trial in stage 1V melanoma patients with MK2-23. In 89percent of patient's post-immunization sera reacted with Colo 38melanoma cells, and inhibited binding of 763.74 to Colo 38 cellssuggesting induction of Ab3 antibodies. In 6 of 15 patients there was areduction in size of metastatic lesions reported but study details werenot furnished. The specificity of the antibodies in patient sera waspartially characterized and it is unclear whether Ab3 antibodies, to theextent that they were present, were responsible for any of the clinicalresponse observed, since the 763.74 antibody did not have innateanti-tumor effects. U.S. Pat. No. 5,866,124 teaches the chimericanti-idiotypic antibody MK2-CHγ1, and its derivatives, directed againstan anti-HMW-MAA antibody, 763.74, derived from MK2-23.

A number of inventions, such as U.S. Pat. No. 5,017,693, U.S. Pat. No.5,707,603, U.S. Pat. No. 5,817,774, U.S. Pat. No. 6,248,870, U.S. Pat.No. 5,112,954, U.S. Pat. No. 6,238,667, teach conjugating compounds toantibodies directed against HMW-MAA but fail to disclose their utilityin treatment of cancerous disease. Importantly, were these antibodieseffective as anti-cancer therapies alone, they would not require aconjugate to impart either cytotoxic or cytostastic effects.

These patents and patent applications identify MCSP antigens and relatedantibodies but fail to disclose the isolated monoclonal antibody of theinstant invention, or to teach or suggest the utility of the isolatedmonoclonal antibody of the instant invention.

SUMMARY OF THE INVENTION

The instant inventors have previously been awarded U.S. Pat. No.6,180,357, entitled “Individualized Patient Specific Anti-CancerAntibodies” directed to a process for selecting individually customizedanti-cancer antibodies, which are useful in treating a cancerousdisease. For the purpose of this document, the terms “antibody” and“monoclonal antibody” (mAb) may be used interchangeably and refer tointact immunoglobulins produced by hybridomas (e.g. murine or human),immunoconjugates and, as appropriate, immunoglobulin fragments andrecombinant proteins derived from said immunoglobulins, such as chimericand humanized immunoglobulins, F(ab′) and F(ab′)₂ fragments,single-chain antibodies, recombinant immunoglobulin variable regions(Fv)s, fusion proteins etc. It is well recognized in the art that someamino acid sequence can be varied in a polypeptide without significanteffect on the structure or function of the protein. In the molecularrearrangement of antibodies, modifications in the nucleic or amino acidsequence of the backbone region can generally be tolerated. Theseinclude, but are not limited to, substitutions (preferred areconservative substitutions), deletions or additions. Furthermore, it iswithin the purview of this invention to conjugate standardchemotherapeutic modalities, e.g. radionuclides, with the CDMAB of theinstant invention, thereby focusing the use of said chemotherapeutics.The CDMAB can also be conjugated to toxins, cytotoxic moieties, enzymese.g. biotin conjugated enzymes, or hematogenous cells, thereby formingantibody conjugates.

This application utilizes the method for producing patient specificanti-cancer antibodies as taught in the '357 patent for isolatinghybridoma cell lines which encode for cancerous disease modifyingmonoclonal antibodies. These antibodies can be made specifically for onetumor and thus make possible the customization of cancer therapy. Withinthe context of this application, anti-cancer antibodies having eithercell-killing (cytotoxic) or cell-growth inhibiting (cytostatic)properties will hereafter be referred to as cytotoxic. These antibodiescan be used in aid of staging and diagnosis of a cancer, and can be usedto treat tumor metastases.

The prospect of individualized anti-cancer treatment will bring about achange in the way a patient is managed. A likely clinical scenario isthat a tumor sample is obtained at the time of presentation, and banked.From this sample, the tumor can be typed from a panel of pre-existingcancerous disease modifying antibodies. The patient will beconventionally staged but the available antibodies can be of use infurther staging the patient. The patient can be treated immediately withthe existing antibodies and/or a panel of antibodies specific to thetumor can be produced either using the methods outlined herein orthrough the use of phage display libraries in conjunction with thescreening methods herein disclosed. All the antibodies generated will beadded to the library of anti-cancer antibodies since there is apossibility that other tumors can bear some of the same epitopes as theone that is being treated. The antibodies produced according to thismethod may be useful to treat cancerous disease in any number ofpatients who have cancers that bind to these antibodies.

Using substantially the process of U.S. Pat. No. 6,180,357, and asdisclosed in Ser. No. 10/348,231, the mouse monoclonal antibody11BD-2E11-2 was obtained following immunization of mice with cells froma patient's breast tumor biopsy. The 11BD-2E11-2 antigen was expressedon the cell surface of several human cell lines from different tissueorigins. The breast cancer cell line MCF-7 and ovarian cancer cell lineOVCAR-3 were susceptible to the cytotoxic effects of 11BD-2E11-2 invitro.

The result of 11BD-2E11-2 cytotoxicity against MCF-7 and OVCAR-3 cellsin culture was further extended by its anti-tumor activity towards thesecancer cells when transplanted into mice (as disclosed in Ser. No.10/762,129). Pre-clinical xenograft tumor models are considered validpredictors of therapeutic efficacy.

In a preventative in vivo model of human breast cancer, 11BD-2E11-2prevented tumor growth and reduced tumor burden (as disclosed in Ser.No. 10/762,129). At day 51 (soon after last treatment), the mean tumorvolume in the 11BD-2E11-2 treated group was 20 percent of the isotypecontrol. Monitoring continued past 280 days post-treatment. 40 percentof the 11BD-2E11-2 treatment group was still alive at over 7.5 monthspost-implantation. Conversely, the isotype control group had 100 percentmortality after 6.5 months post-treatment. Therefore 11BD-2E11-2enhanced survival and decreased the tumor burden compared to thecontrol-treated groups in a well-established model of human breastcancer.

To determine if 11BD-2E11-2 was efficacious in more than one model ofhuman breast cancer, its anti-tumor activity against MDA-MB-468 (MB-468)cells in an established model of breast cancer was determined (asdisclosed in Ser. No. 10/810,744). 11BD-2E11-2 reduced tumor growth by25 percent in comparison to the buffer control. Therefore, 11BD-2E11-2was effective in preventing tumor growth in an established as well as apreventative breast cancer xenograft model. In addition, 11BD-2E11-2displayed anti-tumor activity in at least two different models of breastcancer.

In addition to the beneficial effects in a model of human breast cancer,11BD-2E11-2 treatment also had anti-tumor activity against OVCAR-3 cellsin a preventative ovarian cancer model (as disclosed in Ser. No.10/762,129). In this model, body weight was used as a surrogate measureof tumor progression. At day 80 post-implantation (16 days after the endof treatment) the mice in the treated group had 87.6 percent the meanbody weight of the control group (p=0.015). Thus, 11BD-2E11-2 treatmentwas efficacious as it delayed tumor progression compared to the buffercontrol treated group in a well-established model of human ovariancancer. The anti-tumor activities of 11BD-2E11-2, in several differentcancer models, make it an attractive anti-cancer therapeutic agent.

To determine if 11BD-2E11-2 was efficacious in more than one model ofhuman ovarian cancer, its anti-tumor activity against ES-2+SEAP cells(ES-2 ovarian cancer cells transfected with human placental secretedalkaline phosphatase (SEAP)) in an established model of ovarian cancerwas determined (as disclosed in Ser. No. 10/810,744). 11BD-2E11-2enhanced survival in a cohort of mice in the treatment group incomparison to buffer control. In addition, 1 mouse within the11BD-2E11-2 treatment group displayed greatly reduced circulating SEAPlevels after treatment. Circulating SEAP levels can be used as anindicator of tumor burden. Therefore, 11BD-2E11-2 was effective inpreventing tumor growth in an established as well as a preventativeovarian cancer xenograft model. In addition, 11BD-2E11-2 displayedanti-tumor activity in two different models of human ovarian cancer.

Biochemical data indicated that the antigen for 11BD-2E11-2 is MCSP (asdisclosed in Ser. No. 10/810,744) and previous immunohistochemicalanalysis and in vitro studies performed in other laboratories havedemonstrated the expression of MCSP on melanoma cells and have indicateda role for MCSP in tumor adhesion, invasion and metastasis.Consequently, the efficacy of 11BD-2E11-2 was determined in both apreventative and established model of human melanoma. In thepreventative model of melanoma, on day 55 (5 days after the end oftreatment), the mean tumor volume in the 11BD-2E11-2 treated group was58 percent of the buffer control treated group (p=0.046). In theestablished model, the antibody 11BD-2E11-2 suppressed tumor growth by49 percent in comparison to the buffer control treated group after thetreatment period. The result did not reach significance (p=0.1272) dueto the limited number of animals in this experiment, but the trend wasclear. Therefore, 11BD-2E11-2 was effective in preventing tumor growthin an established as well as a preventative melanoma cancer xenograftmodel. In addition, 11BD-2E11-2 displayed anti-tumor activity in twodifferent models of human breast and ovarian cancer and in a humanmelanoma model.

In order to validate the 11BD-2E11-2 epitope as a drug target, theexpression of 11BD-2E11-2 antigen in frozen normal human tissues wasdetermined (as disclosed in Ser. No. 10/810,744). By IHC staining with11BD-2E11-2, the majority of the tissues failed to express the 1BD-2E11-2 antigen, including the cells of the vital organs, such as theliver, kidney and heart. Albeit, there was staining to the smooth musclefibers of blood vessels in almost all of the tissues. There was alsoepithelial staining for some of the tissues.

Localization of the 11BD-2E11-2 antigen and its prevalence within breastcancer patients is important in assessing the benefits of 11BD-2E11-2immunotherapy to patients and designing effective clinical trials. Toaddress 11BD-2E11-2 antigen expression in breast tumors from cancerpatients, tumor tissue samples from 8 (7 additional samples werecompletely detached or not representative of the tumor on the microarrayslide) individual breast cancer patients were screened for expression ofthe 11BD-2E11-2 antigen (as disclosed in Ser. No. 10/810,744). Theresults of the study showed that 62 percent of tissue samples positivelystained for the 11BD-2E11-2 antigen. Expression of 11BD-2E11-2 withinpatient samples appeared specific for cancer cells as staining wasrestricted to malignant cells. In addition, 11BD-2E11-2 stained 0 of 3(2 additional samples again were completely detached from the microarrayslide) samples of normal tissue from breast cancer patients. When tumorswere analyzed based on their stage, or degree to which the canceradvanced, results did not suggest a trend towards greater positiveexpression with higher tumor stage for 11BD-2E11-2. However, the resultwas limited by the small sample size.

Localization of the 11BD-2E11-2 antigen and its prevalence withinmelanoma cancer patients population was determined because the antigenfor 11BD-2E11-2 is MCSP (as disclosed in Ser. No. 10/810,744) and thatprevious immunohistochemical analysis and in vitro studies performed inother laboratories have demonstrated the expression of MCSP on melanomacells. This is important in assessing the benefits of 11BD-2E11-2immunotherapy for melanoma patients and designing effective clinicaltrials. To address 11BD-2E11-2 antigen expression in melanoma tumorsfrom cancer patients, tumor tissue samples from 33 individual melanomacancer patients were assessed for expression of the 11BD-2E11-2 antigen.The results of the study showed that 67 percent of tissue samplesstained positively for the 11BD-2E11-2 antigen. Expression of11BD-2E11-2 within patient samples appeared specific for cancer cells asstaining was restricted to malignant cells. In addition, 11BD-2E11-2stained 0 of 6 available samples of normal tissue from melanoma cancerpatients.

Biochemical data indicate that the antigen recognized by 11BD-2E11-2 isMCSP (as disclosed in Ser. No. 10/810,744). This was supported bystudies showing that 11BD-2E11-2 immunoprecipitated protein wasrecognized by an antibody to the rat homologue of MCSP, and thatanti-MCSP immunoprecipitated protein was recognized by 11BD-2E11-2.These IHC and biochemical results demonstrate that 11BD-2E11-2 bound tothe MCSP antigen. Thus, the preponderance of evidence showed that11BD-2E11-2 mediated anti-cancer effects through ligation of an uniqueepitope present on MCSP. Additional biochemical data, as outlinedherein, also demonstrate that the antigen recognized by 11BD-2E11-2 isMCSP. These antibody epitope mapping results indicated that 11BD-2E11-2may bind to a discontinuous epitope with two major binding sites.

In toto, this data demonstrates that the 11BD-2E11-2 antigen is a cancerassociated antigen and is expressed in humans, and is a pathologicallyrelevant cancer target. Further, this data also demonstrates the bindingof the 11BD-2E11-2 antibody to human cancer tissues, and can be usedappropriately for assays that can be diagnostic, predictive of therapy,or prognostic. In addition, the cell localization of this antigen isindicative of the cancer status of the cell due to the lack ofexpression of the antigen in most non-malignant cells, and thisobservation permits the use of this antigen, its gene or derivatives,its protein or its variants to be used for assays that can bediagnostic, predictive of therapy, or prognostic.

A number of distinct anti-MCSP antibodies have been developed and testedin many in vitro and in vivo systems. In pre-clinical models, with theexception of one study that was not reproduced, naked anti-MCSPantibodies have been shown to be ineffective in tumor reduction orenhancement of survival in several different melanoma models and oneglioma model; other cancer types have not been studied with anti-MCSPantibodies. All trials of naked anti-MCSP antibodies in humans havefailed to result in any positive clinical outcomes. Naked 11BD-2E11-2has been shown to enhance survival and decrease tumor burden in murinemodels of human breast cancer. 11BD-2E11-2 has also inhibited tumorprogression and enhanced survival in murine models of human ovariancancer. Anti-MCSP antibodies have been conjugated to numerous toxic orchemotherapeutic agents, and these conjugates have demonstrated positivein vivo results when tested in murine models of melanoma. There havebeen no reports of anti-MCSP conjugates tested in humans, so the safetyof these conjugates is not known. Delivery of monoclonal antibody alonehowever has been well tolerated with little, if any associated toxicity.Therefore if treatment of a cancer patient with a naked anti-MCSPantibody could result in a positive clinical outcome, it would bebeneficial and an improvement upon what is currently available.Conjugation to a toxic agent is not required for 11BD-2E11-2 to exhibitanti-cancer activity; therefore the specific safety concerns associatedwith administration of antibody-toxin conjugate are not applicable. Manyanti-MCSP antibodies have also been used to generate anti-idiotypicantibodies, which have been tested in both animals and humans. In smallnon-blinded trials, when the immunization of patients withanti-idiotypic antibodies resulted in a detectable anti-MCSP immuneresponse, there was an increase in median survival of these patientscompared to patients who did not develop a specific immune response. Inthe examples given, targeting MCSP to obtain a positive clinicalresponse may result through the administration of anti-idiotypicantibodies. A problem with this approach is that not all patients whowere immunized with the anti-idiotypic antibodies developed an anti-MCSPresponse. Therefore if an anti-MCSP antibody were available that couldresult in positive clinical outcomes upon direct administration, thiswould overcome the problem of relying on a patient's own immune responsefor producing a clinical benefit. 11BD-2E11-2 is such an antibody as itdirectly targets MCSP and exhibits anti-cancer effects in pre-clinicalxenograft tumor models, which are considered valid predictors oftherapeutic efficacy.

In all, this invention teaches the use of the 1 BD-2E11-2 antigen as atarget for a therapeutic agent, that when administered can reduce thetumor burden (thereby delaying disease progression) of a cancerexpressing the antigen in a mammal, and can also lead to a prolongedsurvival of the treated mammal. This invention also teaches the use of aCDMAB (11BD-2E11-2), and its derivatives, to target its antigen toreduce the tumor burden of a cancer expressing the antigen in a mammal,and to prolong the survival of a mammal bearing tumors that express thisantigen. Furthermore, this invention also teaches the use of detectingthe 11BD-2E11-2 antigen in cancerous cells that can be useful for thediagnosis, prediction of therapy, and prognosis of mammals bearingtumors that express this antigen.

If a patient is refractory to the initial course of therapy ormetastases develop, the process of generating specific antibodies to thetumor can be repeated for re-treatment. Furthermore, the anti-cancerantibodies can be conjugated to red blood cells obtained from thatpatient and re-infused for treatment of metastases. There have been feweffective treatments for metastatic cancer and metastases usuallyportend a poor outcome resulting in death. However, metastatic cancersare usually well vascularized and the delivery of anti-cancer antibodiesby red blood cells can have the effect of concentrating the antibodiesat the site of the tumor. Even prior to metastases, most cancer cellsare dependent on the host's blood supply for their survival andanti-cancer antibodies conjugated to red blood cells can be effectiveagainst in situ tumors as well. Alternatively, the antibodies may beconjugated to other hematogenous cells, e.g. lymphocytes, macrophages,monocytes, natural killer cells, etc.

There are five classes of antibodies and each is associated with afunction that is conferred by its heavy chain. It is generally thoughtthat cancer cell killing by naked antibodies are mediated either throughantibody-dependent cell-mediated cytotoxicity (ADCC) orcomplement-dependent cytotoxicity (CDC). For example murine IgM andIgG2a antibodies can activate human complement by binding the C-1component of the complement system thereby activating the classicalpathway of complement activation which can lead to tumor lysis. Forhuman antibodies, the most effective complement-activating antibodiesare generally IgM and IgG1. Murine antibodies of the IgG2a and IgG3isotype are effective at recruiting cytotoxic cells that have Fcreceptors which will lead to cell killing by monocytes, macrophages,granulocytes and certain lymphocytes. Human antibodies of both the IgG1and IgG3 isotype mediate ADCC.

Another possible mechanism of antibody-mediated cancer killing may bethrough the use of antibodies that function to catalyze the hydrolysisof various chemical bonds in the cell membrane and its associatedglycoproteins or glycolipids, so-called catalytic antibodies.

There are two additional mechanisms of antibody-mediated cancer cellkilling which are more widely accepted. The first is the use ofantibodies as a vaccine to induce the body to produce an immune responseagainst the putative antigen that resides on the cancer cell. The secondis the use of antibodies to target growth receptors and interfere withtheir function or to down regulate that receptor so that its function iseffectively lost.

The clinical utility of a cancer drug is based on the benefit of thedrug under an acceptable risk profile to the patient. In cancer therapysurvival has generally been the most sought after benefit, however thereare a number of other well-recognized benefits in addition to prolonginglife. These other benefits, where treatment does not adversely affectsurvival, include symptom palliation, protection against adverse events,prolongation in time to recurrence or disease-free survival, andprolongation in time to progression. These criteria are generallyaccepted and regulatory bodies such as the U.S. Food and DrugAdministration (F.D.A.) approve drugs that produce these benefits(Hirschfeld et al. Critical Reviews in Oncology/Hematology 42:137-1432002). In addition to these criteria it is well recognized that thereare other endpoints that may presage these types of benefits. In part,the accelerated approval process granted by the U.S. F.D.A. acknowledgesthat there are surrogates that will likely predict patient benefit. Asof year-end (2003), there have been sixteen drugs approved under thisprocess, and of these, four have gone on to full approval, i.e.,follow-up studies have demonstrated direct patient benefit as predictedby surrogate endpoints. One important endpoint for determining drugeffects in solid tumors is the assessment of tumor burden by measuringresponse to treatment (Therasse et al. Journal of the National CancerInstitute 92(3):205-216 2000). The clinical criteria (RECIST criteria)for such evaluation have been promulgated by Response EvaluationCriteria in Solid Tumors Working Group, a group of international expertsin cancer. Drugs with a demonstrated effect on tumor burden, as shown byobjective responses according to RECIST criteria, in comparison to theappropriate control group tend to, ultimately, produce direct patientbenefit. In the pre-clinical setting tumor burden is generally morestraightforward to assess and document. In that pre-clinical studies canbe translated to the clinical setting, drugs that produce prolongedsurvival in pre-clinical models have the greatest anticipated clinicalutility. Analogous to producing positive responses to clinicaltreatment, drugs that reduce tumor burden in the pre-clinical settingmay also have significant direct impact on the disease. Althoughprolongation of survival is the most sought after clinical outcome fromcancer drug treatment, there are other benefits that have clinicalutility and it is clear that tumor burden reduction, which may correlateto a delay in disease progression, extended survival or both, can alsolead to direct benefits and have clinical impact (Eckhardt et al.Developmental Therapeutics: Successes and Failures of Clinical TrialDesigns of Targeted Compounds; ASCO Educational Book, 39^(th) AnnualMeeting, 2003, pages 209-219).

Accordingly, it is an objective of the invention to utilize a method forproducing cancerous disease modifying antibodies from cells derived froma particular individual which are cytotoxic with respect to cancer cellswhile simultaneously being relatively non-toxic to non-cancerous cells,in order to isolate hybridoma cell lines and the corresponding isolatedmonoclonal antibodies and antigen binding fragments thereof for whichsaid hybridoma cell lines are encoded.

It is an additional objective of the invention to teach CDMAB andantigen binding fragments thereof.

It is a further objective of the instant invention to produce CDMABwhose cytotoxicity is mediated through ADCC.

It is yet an additional objective of the instant invention to produceCDMAB whose cytotoxicity is mediated through CDC.

It is still a further objective of the instant invention to produceCDMAB whose cytotoxicity is a function of their ability to catalyzehydrolysis of cellular chemical bonds.

A still further objective of the instant invention is to produce CDMABwhich are useful in a binding assay for diagnosis, prognosis, andmonitoring of cancer.

Other objects and advantages of this invention will become apparent fromthe following description wherein, by way of illustration and example,certain embodiments of this invention are set forth.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Western blot of MDA-MB-231 (Lane 1) or OVCAR-3 (Lane 2)membranes probed with 11BD-2E11-2. Membrane proteins were separatedunder reducing conditions. Molecular weight markers are indicated on theright.

FIG. 2. Effect of deglycosylation on the binding of 11BD-2E11-2 toMDA-MB-231 membranes. 11BD-2E11-2 binding to MDA-MB-231 membranes thatwere incubated in deglycosylation buffer only (Lane 1), in a combinationof PNGase F, endo-o-glycosidase, sialidase, galactosidase andglucosaminodase (Lane 2), in a combination of PNGase, endo-o-glycosidaseand sialidase (Lane 3), in sialidase only (Lane 4), inendo-o-glycosidase only (Lane 5), and in PNGase only (Lane 6).

FIG. 3. SDS-PAGE (Panel A) and Western blot (Panel B) of MDA-MB-231membrane proteins immunoprecipitated with 11BD-2E11-2. Lane 1 representsthe molecular weight standard, Lane 2 the MDA-MB-231 membrane proteins,Lane 3 the 11BD-2E11-2 immunoprecipitated material and Lane 4 theisotype control immunoprecipitated material.

FIG. 4. Western blots of proteins probed with 11BD-2E11-2 (Panel A),IgG1 isotype control (clone 107.3, Panel B), anti-rat NG2 (polyclonal,Panel C), normal rabbit IgG (Panel D), anti-MCSP (clone 9.2.27, Panel E)and IgG2a isotype control (clone G155-228, Panel F). Lane 1: 11BD-2E11-2immunoprecipitate, Lane 2: IgG1 isotype control (clone 107.3)immunoprecipitate, Lane 3: anti-MCSP (clone 9.2.27) immunoprecipitate,Lane 4: IgG2a isotype control (clone G155-228) immunoprecipitate, Lane5: MDA-MB-231 membranes and Lane 6: sample buffer only (negativecontrol).

FIG. 5. Intensity of binding (Boehringer light units) of 11BD-2E11-2-HRPto MCSP peptide array.

FIG. 6. Representative FACS histograms of 11BD-2E11-2, isotype controlor anti-EGFR directed against several cancer cell lines and non-cancercells.

FIG. 7. Representative micrographs showing the binding pattern obtainedwith 11BD-2E11-2 (A) and the isotype control antibody (B) on tissuessections of heart from a frozen normal human tissue array. There is nostaining of 11BD-2E11-2 to cardiac muscle fibers. Magnification is 200×.

FIG. 8. Representative micrographs showing the binding pattern obtainedwith 11BD-2E11-2 (A), anti-actin (B) and the isotype control antibody(C) on tissues sections of skeletal muscle from a frozen normal humantissue array. 11BD-2E11-2 did not stain skeletal muscle but there isstaining to the smooth muscles of blood vessels (arrow). Magnificationis 200×.

FIG. 9. Representative micrograph of 11BD-2E11-2 (A) and isotype controlantibody (B) binding to breast cancer tumor (infiltrating ductcarcinoma). The black arrow in panel A points to tumor cells.Magnification is 200×.

FIG. 10. Representative micrographs showing the binding pattern obtainedwith 11BD-2E11-2 (A), positive control anti-CD63 (NKI-C3) (B) and thenegative isotype control antibody (C) on tissues sections of malignantmelanoma from a frozen melanoma human tissue array. Magnification is200×.

FIG. 11. Representative micrographs showing the binding pattern obtainedwith 11BD-2E11-2 on malignant melanoma (A) and normal skin (B) tissuessections from a frozen melanoma human tissue array. There is strongstaining of 11BD-2E11-2 to the malignant melanoma but not to the normalskin. Magnification is 200×.

FIG. 12. Effect of 11BD-2E11-2 or buffer control on tumor growth in apreventative MDA-MB-468 breast cancer model. The dashed line indicatesthe period during which the antibody was administered. Data pointsrepresent the mean+/−SEM.

FIG. 13. Survival of tumor-bearing mice after treatment with 11BD-2E11-2or buffer control antibody in an established ES-2 xenograft study.

FIG. 14. SEAP levels of tumor-bearing mice before, during and aftertreatment with 11BD-2E11-2 or buffer control in an established ES-2xenograft study.

FIG. 15. Effect of 11BD-2E11-2 or buffer control on tumor growth in apreventative A2058 melanoma cancer model. The dashed line indicates theperiod during which the antibody was administered. Data points representthe mean+/−SEM.

FIG. 16. Effect of 11BD-2E11-2 or buffer control on tumor growth in anestablished A2058 melanoma cancer model. The dashed line indicates theperiod during which the antibody was administered. Data points representthe mean+/−SEM.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Identification ofBinding Proteins by Western Blotting

To identify the antigen(s) recognized by the antibody 11BD-2E11-2, cellmembranes expressing this antigen were subjected to gel electrophoresisand transferred using Western blotting to membranes to determine theproteins detected by this antibody (as disclosed in Ser. No.10/810,744).

1. Membrane Preparation

Previous work demonstrated binding by FACS of 11BD-2E11-2 to the breastcancer line MDA-MB-231 (MB-231). Previous work also demonstrated11BD-2E11-2 efficacy against the ovarian cancer cell line OVCAR-3.Accordingly, membrane preparations from these 2 cell lines were used forantigen identification. Additional Western blotting andimmunoprecipitation studies have also demonstrated a similar bindingpattern of 11BD-2E11-2 to A2058 membrane preparations.

Total cell membranes were prepared from confluent cultures of MB-231breast cancer or OVCAR-3 ovarian cells. Media was removed from cellstacks and the cells were washed with phosphate buffered saline. Cellswere dissociated with dissociation buffer (Gibco-BRL, Grand Island,N.Y.) for 20 min at 37° C. on a platform shaker. Cells were collectedand centrifuged at 900 g for 10 min at 4° C. After centrifugation, cellpellets were resuspended in PBS and centrifuged again at 900 g for 10min at 4° C. to wash. Pellets were stored at −80° C. Cell pellets wereresuspended in homogenization buffer containing 1 tablet per 50 mL ofComplete protease inhibitor cocktail (Roche, Laval QC) at a ratio of 3mL buffer per gram of cells. The cell suspension was subjected tohomogenization using a polytron homogenizer on ice in order to lyse thecells. The cell homogenate was centrifuged at 15,000 g for 10 min at 4°C. to remove the nuclear particulate. Supernatant was harvested, dividedinto tubes and then centrifuged at 75,600 g for 90 min at 4° C.Supernatant was carefully removed from the tubes and each membranepellet was resuspended in approximately 5 mL homogenization buffer. Theresuspended pellets from all tubes were combined together in one tubeand centrifuged at 75,600 g for 90 min at 4° C. Supernatant from thetubes was carefully removed, and the pellets were weighed.Solubilization buffer containing 1 percent Triton X-100 was added to thepellets at a ratio of 3 mL buffer per gram of membrane pellet. Membraneswere solubilized by shaking on a platform shaker at 300 rpm for 1 hr onice. The membrane solution was centrifuged at 75,600 g to pelletinsoluble material. The supernatant containing the solubilized membraneproteins was carefully removed from tubes, assayed for protein content,and stored at −80° C.

2. SDS-PAGE and Western Blot

Membrane proteins were separated by SDS-polyacrylamide gelelectrophoresis. 20 μg of membrane protein was mixed with SDS-PAGEsample buffer containing 100 mM DTT and was loaded onto a lane of an 8percent SDS-PAGE gel. A sample of prestained molecular weight markers(Invitrogen, Burlington, ON) was run in a reference lane.Electrophoresis was carried out at 100 V for 10 minutes, followed by 150V until sufficient resolution of the prestained molecular weight markerswas observed. Proteins were transferred from the gel to PVDF membranes(Millipore, Billerica, Mass.) by electroblotting for 16 hr at 40 V.Transfer was assessed by noting complete transfer of the prestainedmarkers from the gel to the membrane. Following transfer, membranes wereblocked with 5 percent skim milk powder in Tris-buffered salinecontaining 0.5 percent Tween-20 (TBST) for 2 hr. Membranes were washedonce with TBST and then incubated with 5 μg/mL 11BD-2E11-2 diluted in 3percent skim milk powder in TBST for 2 hr. After washing 3 times withTBST, membranes were incubated with goat anti-mouse IgG (Fc) conjugatedto horseradish peroxidase (HRP) from Jackson Immunologicals (West GrovePa.). This incubation was followed by washing 3 times with TBST,followed by incubation with the HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (substrate kit from Vector Laboratories, Burlington ON).

In FIG. 1, 11BD-2E11-2 clearly binds to 3 molecular weight regions ofthe separated MB-231 (Lane 1) and OVCAR-3 (Lane 2) membrane proteins. Bycomparison to the molecular weight (MW) standards, the antibody binds toproteins of MW approximately 150, 240 and 280 kDa. All further studieswere done using the MB-231 membranes since stronger reactivity was seenwith this cell line.

EXAMPLE 2 Determining Glycosylation of Antigens Bound by 11BD-2E11-2

In order to determine if the antigen(s) recognized by the antibody11BD-2E11-2 were glycoproteins, MB-231 membranes were incubated withdifferent combinations of PNGase F, endo-o-glycosidase, sialidase,galactosidase and glucosaminidase. Membranes were separated by SDS-PAGEfollowed by Western blotting as described with 1 BD-2E11-2. FIG. 2demonstrates the result of 11BD-2E11-2 binding to MB-231 membranes thatwere incubated in deglycosylation buffer only (Lane 1), in a combinationof PNGase F, endo-o-glycosidase, sialidase, galactosidase andglucosaminodase (Lane 2), in a combination of PNGase, endo-o-glycosidaseand sialidase (Lane 3), in sialidase only (Lane 4), inendo-o-glycosidase only (Lane 5), and in PNGase only (Lane 6). Treatmentof MB-231 membranes with glycosidases does not eliminate binding of11BD-2E11-2, however a molecular weight shift of the proteins isobserved in all lanes, indicating that the antigen recognized by11BD-2E11-2 was a glycoprotein.

EXAMPLE 3 Identification of Antigens Bound by 11BD-2E11-2 1.Immunoprecipitation

The identification of the antigen for 111BD-2E11-2 was carried out byisolating the cognate ligand through immunoprecipitation of solublizedmembrane gylcoproteins with the antibody. 100 μL of Protein G Dynabeads(Dynal Biotech, Lake Success N.Y.) were washed 3 times with 1 mL of 0.1M sodium phosphate buffer pH 6.0. 100 μg of 11BD-2E11-2 in a totalvolume of 100 μL 0.1 M sodium phosphate buffer pH 6.0 was added to thewashed beads. The mixture was incubated for 1 hr with rotational mixing.Unbound antibody was removed and the 11BD-2E11-2 coated beads werewashed 3 times with 0.5 mL 0.1 M sodium phosphate pH 7.4 containing 0.1percent Tween-20. The 11BD-2E11-2 coated beads were washed 2 times with1 mL 0.2 M triethanolamine pH 8.2. 11BD-2E11-2 was chemicallycrosslinked to the beads by adding 1 mL of 0.02 M dimethylpimelimidatein 0.2 M triethanolamine pH 8.2 and incubating with rotational mixingfor 30 min. The reaction was stopped by incubating the beads with 1 mLof 0.05 M Tris pH 7.5, for 15 min with rotational mixing. The11BD-2E11-2 crosslinked beads were washed 3 times with 1 mL of 1 mMKH₂PO₄, 10 mM Na₂HPO₄, 137 mM NaCl, 2.7 mM KCl (PBS) containing 0.1percent Tween-20. The 11BD-2E11-2 crosslinked beads were pre-eluted byincubation with 0.1 M citrate pH 3.0 for 3 min followed by 3 washes in0.1 M PBS containing 0.1 percent Tween-20. A second set of antibodycrosslinked beads were prepared in the same manner described using amouse IgG₁ antibody (clone 107.3 from BD Biosciences, Oakville ON) totrinitrophenol, an irrelevant molecule, which was used as a negativeIgG₁ isotype control.

The 11BD-2E11-2 crosslinked beads were blocked by incubating in 1percent BSA in 0.1 M sodium phosphate pH 7.4 with rotational mixing for30 minutes at 4° C. The beads were washed 3 times with 0.1 M sodiumphosphate pH 7.4. 500 μg of total membrane preparation from MB-231 cellswas incubated with the 11BD-2E11-2 crosslinked beads with rotationalmixing for 2.5 hr at 4° C. The immunocomplex bound beads were washedthree times with 1 mL of 1 mM KH₂PO₄, 10 mM Na₂HPO₄, 287 mM NaCl, 2.7 mMKCl containing 1 percent Triton X-100. 11BD-2E11-2 bound protein waseluted from the 11BD-2E11-2 crosslinked beads by incubation with 30 μLof 0.1 M citrate pH 3.0 for 3 min with gentle mixing. The eluted proteinwas brought to neutral pH by the addition of 9 μL of 1 M Tris pH 9. Theneutralized eluted protein was stored at −80° C. The 11BD-2E11-2crosslinked beads were washed with 3 mL PBS containing 0.1 percentTween-20. The IgG₁ isotype control (clone 107.3) crosslinked beads wereincubated with MB-231 membrane proteins and processed in the same manneras the 11BD-2E11-2 beads.

Two batches of 11BD-2E11-2 immunoprecipitated protein from MB-231membrane proteins were produced as described and combined together. Thesame was done for the IgG1 (clone 107.3) isotype control beads.Sixty-two percent of this immunoprecipitate mixture (corresponding tothe amount of protein immunoprecipitated from 620 μg of MB-231 membraneproteins) was loaded onto a single lane of a 4-20 percent gradientSDS-PAGE gel. The same amount of material produced from the 107.3crosslinked beads was loaded in an adjacent lane, as was 20 μg of MB-231membrane proteins. A sample of unstained molecular weight markers(Invitrogen, Burlington ON) or pre-stained molecular weight markers wererun in reference lanes. The sample was separated by electrophoresis at100 V for 10 min, followed by 150 V for 60 minutes. Proteins werestained by incubating the gel in SYPRO Ruby™ (BioRad, Mississauga, ON).In a parallel Western blot, 18 percent of the immunoprecipitate mixture,which corresponded to the amount of protein immunoprecipitated from 180μg of MB-231 membrane proteins, and the same amount of material producedfrom the IgG1 isotype control (clone 107.3) crosslinked beads, wereseparated by electrophoresis. Proteins were transferred from the gel toPVDF membranes (Millipore, Billerica, Mass.) by electroblotting for 16hr at 40 V. After transfer, the membrane was blocked with 5 percent skimmilk powder in TBST for 2 hr. The membrane was probed with 5 μg/mL11BD-2E11-2 diluted in 3 percent skim milk powder in TBST for 2 hr.After washing 3 times with TBST, the membrane was incubated with goatanti-mouse IgG (Fc) conjugated HRP for 1 hr. This incubation wasfollowed by washing 3 times with TBST, followed by incubation with theHRP substrate TMB.

FIG. 3 depicts the gel and Western blot obtained from the proteinsimmunoprecipitated by 11BD-2E11-2. On the gel (Panel A) Lane 1represents the molecular weight standard and Lane 2 represents theMB-231 membrane proteins. There were two distinct bands of MW 240 and280 kDa in the lane containing the 11BD-2E11-2 immunoprecipitatedmaterial (Lane 3) that were not present in the lane containing the 107.3immunoprecipitated material (Lane 4). On the corresponding Western blot(Panel B), 11BD-2E11-2 reacts strongly with the 11BD-2E11-2immunoprecipitated proteins of MW 240 and 280 kDa (Lane 3). On theWestern blot 11BD-2E11-2 also reacts strongly to an additional band inthe 11BD-2E11-2 immunoprecipitated protein at 150 kDa; this band was notdetectable on the stained gel. The reactivity profile of 11BD-2E11-2 to11BD-2E11-2 immunoprecipitated protein was similar to that seen in theMB-231 total membranes (Lane 2). There was no reactivity of 11BD-2E11-2to proteins immunoprecipitated by IgG1 isotype control (clone 107.3;Lane 4), indicating that the binding of 11BD-2E11-2 to theimmunoprecipitated protein was specific, and not due to the presence ofcontaminating proteins.

2. Mass Spectrometry

The regions of the gel corresponding to the 240 and 280 kDa proteinimmunoprecipitated by 11BD-2E11-2 (FIG. 3, Panel A, Lane 3) were cut outusing sterile scalpels. These gel slices were then used foridentification of proteins by mass spectrometry using MALDI/MS andLC/MS/MS.

The samples were subjected to proteolytic digestion on a PROGESTworkstation using trypsin, and a portion of the resulting digestsupernatant was used for MALDI/MS analysis. Spotting was performedrobotically (ProMS) with ZipTips; peptides were eluted form the C18material with matrix (α-cyano 4-hydroxy cinnamic acid) prepared in 60percent acetonitrile, 0.2 percent TFA. MALDI/MS data was acquired on anVoyager DE-STR instrument (Applied Biosystems, Foster City Calif. andthe observed m/z values were submitted to ProFound (Proteometricssoftware package) for peptide mass fingerprint searching. ProFoundqueried a locally stored copy of the NCBInr database. An additionalportion of the digest supernatant was analyzed by nano LC/MS/MS on aMicromass Q-Tof2 using a 75 μm C18 column at a flow-rate of 200 mL/min.MS/MS data were searched using a local copy of MASCOT.

The proteins identified by MALDI/MS and LC/MS/MS are presented in Table1.

TABLE 1 Proteins Identified by 11BD-2E11-2 Immunoprecipitation ofMDA-MB-231 Membranes # of Percent peptides NCBI Sample Observed MWMethod Protein ID coverage matched accession # A 280 kDa MALDI Melanoma-13 20 gi 4503099 associated chondroitin sulfate proteoglycan LC/MS/MSMelanoma 2 gi 34148711 chondroitin sulfate proteoglycan B 240 kDa MALDIMelanoma 14 21 gi 4503099 associated chondroitin sulfate proteoglycanBoth samples were identified as melanoma-associated chondroitin sulfateproteoglycan (MCSP).

3. Confirmation

Confirmation of the putative antigen was carried out by determiningwhether known anti-MCSP antibodies would react with the proteinimmunoprecipitated by 11BD-2E11-2 and vice versa. Immunoprecipitateswere prepared in the same manner as described previously except with theaddition of the mouse anti-MCSP monoclonal antibody 9.2.27 (IgG2a)(Chemicon, Temecula Calif.) and the mouse IgG2a antibody (clone G155-178from BD Biosciences; Oakville ON) to trinitrophenol, an irrelevantmolecule, which was used as a negative IgG2a isotype control.11BD-2E11-2 immunoprecipitate, IgG1 isotype control (clone 107.3)immunoprecipitate, anti-MCSP (clone 9.2.27) immunoprecipitate, IgG2aisotype control (clone G155-228) immunoprecipitate and MB-231 membraneswere separated by SDS-PAGE on six replicate 10 percent gels.Electrophoresis and Western blotting were carried out as describedabove. The membranes were incubated with 5 μg/mL of 11BD-2E11-2, IgG1isotype control (clone 107.3), anti-MCSP (clone 9.2.27), IgG2a isotypecontrol (clone G155-228), rabbit polyclonal anti-rat NG2 antibody (MCSPis the human homologue of rat NG2; Chemicon, Temecula Calif.) and normalrabbit IgG (Sigma, Saint Louis Mo.) diluted in 3 percent skim milkpowder in TBST for 2.5 hr. FIG. 4 demonstrates the results of theWestern blotting as described. FIG. 4 (Panel A) shows the binding of11BD-2E11-2 to 11BD-2E11-2 immunoprecipitate (Lane 1), IgG1 isotypecontrol (clone 107.3) immunoprecipitate (Lane 2), anti-MCSP (clone9.2.27) immunoprecipitate (Lane 3), IgG2a isotype control (cloneG155-228) immunoprecipitate (Lane 4), MB-231 membranes (Lane 5) andsample buffer only (negative control) (Lane 6). 11BD-2E11-2 recognizedthe same three bands of approximately 150, 240 and 280 kDa in both theMB-231 membranes and in the 11BD-2E11-2 immunoprecipitate. Only theupper 280 kDa band was recognized in the anti-MCSP (clone 9.2.27)immunoprecipitate lane. There is no reaction in either of the isotypecontrol immunoprecipitate lanes, indicating that the reactivity of11BD-2E11-2 to the immunoprecipitates was due to proteins beingspecifically bound and immunoprecipitated by both 11BD-2E11-2 and9.2.27. In a parallel blot (Panel B) probed with IgG1 isotype control(clone 107.3), no reactivity was observed in any of the lanes,indicating that the reactivity observed in the blot probed with11BD-2E11-2 was specific. Panel C shows the binding of rabbit polyclonalanti-rat NG2 antibody to a parallel blot. Anti-NG2 binds to two bands ofapproximately 150 and 240 kDa in the 11BD-2E11-2 immunoprecipitate(Lane 1) while it does not bind to proteins of this molecular weightrange in any of the other lanes. In a parallel blot (Panel D), normalrabbit IgG shows faint non-specific reactivity to proteins in both theIgG2a immunoprecipitate (Lane 4) and MB-231 membranes (Lane 5).Therefore the same reactivity in these lanes on Panel C (probed withrabbit anti-NG2) should be regarded as non-specific. In a parallel blot(Panel E) anti-MCSP (clone 9.2.27) shows only very faint binding to oneband in the anti-MCSP (clone 9.2.27) immunoprecipitate lane (Lane 3,indicated by arrow); this band is not seen in the MB-231 membranes (Lane5) which indicates that 9.2.27 may have a low affinity for this antigenand only show reactivity when it is present in a concentrated form suchas it is in the immunoprecipitated sample. In the final parallel blot(Panel F) probed with IgG2a isotype control (clone G155-228), noreactivity was observed in any of the lanes, indicating that thereactivity observed in the blot probed with anti-MCSP (clone 9.2.27) wasspecific. These results demonstrate that 11BD-2E11-2 immunoprecipitatedprotein was recognized by the rat homologue of MCSP, and that anti-MCSPimmunoprecipitated protein was recognized by 11BD-2E11-2.

The mass spectroscopic identification combined with the confirmationusing known commercial antibodies demonstrates that the antigen for11BD-2E11-2 is MCSP. This is also consistent with the deglycosylationexperiments in Example 2, as the core protein of MCSP is a glycoprotein.

EXAMPLE 4 Antibody Epitope Mapping

Antibody epitope mapping experiments were carried out in order todetermine the region(s) of the MCSP molecule that were recognized by11BD-2E11-2. An overlapping peptide array based on the amino acidsequence of MCSP was synthesized and covalently bound to a cellulosemembrane in a stepwise manner, resulting in a defined arrangement. Eachpeptide was 18 amino acids long with an overlap of 9 amino acids. Thepeptide array was incubated with blocking buffer for several hours.11BD-2E11-2 was conjugated to horseradish peroxidase (HRP) using amodified periodate method following the method of Wilson and Nakane.Following blocking, the peptide array was incubated with 1 μg/mL11BD-2E11-2-HRP in blocking buffer. In a separate experiment, thepeptide array was incubated with a sheep anti-mouse IgG-HRP as anegative control. The peptide array was washed with TBST and incubatedwith a chemiluminescent substrate. The light emitted during thechemiluminescent reaction was quantified for each spot on the peptidearray using a charge coupled device (CCD)-camera, resulting in a signalintensity value (Boehringer light units; BLU) for each peptide. For thisexperiment all signals below 7500 BLU were considered as background. Thebinding data for the peptide array is listed in Table 2 (SEQ ID NOS:1-257, respectively, in order of appearance).

TABLE 2 Binding of 11BD-2E11-2-HRP to MCSP Peptide Array Peptide NumberAmino Acid Sequence BLU 1 MQSGRGPPLPAPGLALAL 566 2 PAPGLALALTLTMLARLA970 3 TLTMLARLASAASFFGEN 11290 4 SAASFFGENHLEVPVATA 494 5HLEVPVATALTDIDLQLQ 905 6 LTDIDLQLQFSTSQPEAL 7196 7 FSTSQPEALLLLAAGPAD937 8 LLLAAGPADHLLLQLYSG 1035 9 HLLLQLYSGRLQVRLVLG 1132 10RLQVRLVLGQEELRLQTP 3383 11 QEELRLQTPAETLLSDSI 1148 12 AETLLSDSIPHTVVLTVV788 13 PHTVVLTVVEGWATLSVD 1069 14 EGWATLSVDGFLNASSAV 1637 15GFLNASSAVPGAPLEVPY 1657 16 PGAPLEVPYGLFVGGTGT 1892 17 GLFVGGTGTLGLPYLRGT2343 18 LGLPYLRGTSRPLRGCLH 1823 19 SRPLRGCLHAATLNGRSL 2035 20AATLNGRSLLRPLTPDVH 1672 21 LRPLTPDVHEGCAEEFSA 4678 22 EGCAEEFSASDDVALGFS5263 23 SDDVALGFSGPHSLAAFP 564 24 GPHSLAAFPAWGTQDEGT 812 25AWGTQDEGTLEFTLTTQS 1943 26 LEFTLTTQSRQAPLAFQA 33781 27RQAPLAFQAGGRRGDFIY 3904 28 GGRRGDFIYVDIFEGHLR 3199 29 VDIFEGHLRAVVEKGQGT2016 30 AVVEKGQGTVLLHNSVFV 1399 31 VLLHNSVPVADGQPHEVS 1114 32ADGQPHEVSVNINAHRLE 1268 33 VHINAHRLEISVDQYPTH 1665 34 ISVDQYPTHTSNRGVLSY1562 35 TSNRGVLSYLEPRGSLLL 2539 36 LEPRGSLLLGGLDAEASR 2576 37GGLDAEASRHLQEHRLGL 1376 38 HLQEHRLGLTPEATNASL 957 39 TPEATNASLLGCMEDLSV4354 40 LGCMEDLSVNGQRRGLRE 5881 41 NGQRRGLREALLTRNMAA 3880 42ALLTRNMAAGCRLEEEEY 3939 43 GCRLEEEEYEDDAYGHYE 731 44 EDDAYGHYEAFSTLAPEA1013 45 AFSTLAPEAWPAMELPEP 844 46 WPAMELPEPCVPEPGLPP 2033 47CVPEPGLPPVFANFTQLL 7330 48 VFANFTQLLTISPLVVAE 2261 49 TISPLVVAEGGTAWLEWR2439 50 GGTAWLEWRHVQPTLDLM 1956 51 HVQPTLDLMEAELRKSQV 2044 52EASLRKSQVLFSVTRGAH 2944 53 LFSVTRGAHYGELELDIL 4346 54 YGELELDILGAQARKMFT3249 55 GAQARKMFTLLDVVNRKA 4077 56 LLDVVNRKARFIHDGSED 3778 57RFIHDGSEDTSDQLVLEV 1287 58 TSDQLVLEVSVTARVPMP 2650 59 SVTARVPMPSCLRRGQTY1327 60 SCLRRGQTYLLPIQVNPV 1342 61 LLPIQVNPVNDPPHIIFP 25 62NDPPHIIFFHGSLMVILE 6 63 HGSLMVILEHTQKPLGPE 564 64 HTQKPLGPEVFQAYDPDS 78165 VFQAYDPDSACEGLTFQV 3015 66 ACEGLTFQVLGTSSCLPV 15941 67LGTSSGLPVERRDQPGEP 2310 68 ERRDQPGEPATEFSCREL 7895 69 ATEFSCRELEAGSLVYVH2724 70 EAGSLVYVHCGGPAQDLT 4799 71 CGGPAQDLTFRVSDGLQA 56703 72FRVSDGLQASPPATLKVV 6138 73 SPPATLKVVAIRPAIQIH 2873 74 AIRPAIQIHRSTGLRLAQ4406 75 RSTGLRLAQGSAMPILPA 4387 76 GSAMPILPANLSVETNAV 2024 77NLSVETNAVGQDVSVLFR 2333 78 GQDVSVLFRVTGALQFGE 4056 79 VTGALQFGELQKHSTGGV1554 80 LQKHSTGGVEGAEWWATQ 962 81 EGAEWWATQAFHQRDVEQ 290 82AFHQRDVEQGRVRYLSTD 1059 83 GRVRYLSTDPQHHAYDTV 842 84 PQHHAYDTVENLALEVQV1173 85 ENLALEVQVGQEILSNLS 3084 86 GQEILSNLSFPVTIQRAT 4928 87FPVTIQRATVWMLRLEPL 2142 88 VWMLRLEPLHTQNTQQET 2345 89 HTQNTQQETLTTAHLEAT2719 90 LTTAHLEATLEEAGPSPP 2513 91 LEEAGPSPPTFHYEVVQA 2380 92TFHYEVVQAPRKGNLQLQ 4209 93 PRKGNLQLQGTRLSDGQG 8990 94 GTRLSDCQGFTQDDIQAG3830 95 FTQDDIQAGRVTYGATAR 4641 96 RVTYGATARASEAVEDTF 1950 97ASEAVEDTFRFRVTAPPY 1463 98 RFRVTAPPYFSPLYTFPI 870 99 FSPLYTFPIHIGGDPDAP1092 100 HIGGDPDAPVLTNVLLVV 1043 101 VLTNVLLVVPEGGEGVLS 169 102PEGGEGVLSADHLFVKSL 640 103 ADHLFVKSLNSASYLYEV 601 104 NSASYLYEVMERPRLGRL2697 105 MERPRLGRLAWRGTQDKT 5728 106 AWRGTQDKTTMVTSFTNE 2771 107TMVTSFTNEDLLRGRLVY 2243 108 DLLRGRLVYQHDDSETTE 2316 109QHDDSETTEDDIPFVATR 3020 110 DDIFFVATRQGESSGDMA 3695 111QGESSGDMAWEEVRGVFR 3949 112 WEEVRGVFRVAIQFVNDH 2674 113VAIQPVNDHAPVQTISRI 4340 114 APVQTISRIFHVARGGRR 6454 115FHVARGGRRLLTTDDVAF 5898 116 LLTTDDVAFSDADSGFAD 1615 117SDADSGFADAQLVLTRKD 1464 118 AQLVLTRKDLLEGSIVAV 1137 119LLFGSIVAVDEPTRPIYR 1972 120 DEPTRPIYRFTQEDLRKR 5531 121FTQEDLRKRRVLFVHSGA 1860 122 RVLFVHSGADRGWIQLQV 465 123DRGWIQLQVSDGQHQATA 812 124 SDGQHQATALLEVQASEP 759 125 LLSVQASEPYLRVANGSS1502 126 YLRVANGSSLVVPQGGQG 4406 127 LVVPQGGQGTIDTAVLHL 1506 128TIDTAVLHLDTNLDIRSG 2535 129 DTNLDIRSGDEVHYHVTA 2159 130DEVHYHVTAGPRWGQLVR 4541 131 GPRWGQLVRAGQPATAFS 9113 132AGQPATAFSQQDLLDGAV 3668 133 QQDLLDGAVLYSHNGSLS 3565 134LYSHNGSLSPEDTMAFSV 3626 135 PEDTMAFSVEAGPVHTDA 2159 136EAGPVHTDATLQVTIALE 1585 137 TLQVTIALEGPLAPLKLV 2444 138GPLAPLKLVRHKKTYVFQ 1100 139 RHKKIYVFQGEAAEIRRD 2108 140GEAAEIRRDQLEAAQEAV 1275 141 QLEAAQEAVPPADIVFSV 902 142PPADIVESVKSPPSAGYL 1224 143 KSPPSAGYLVMVSRGALA 1725 144VMVSRGALADEPPSLDPV 949 145 DEPPSLDPVQSFSQEAVD 1189 146QSFSQEAVDTGRVLYLHS 1447 147 TGRVLYLHSRPEAWSDAF 1661 148RPEAWSDAFSLDVASGLG 2269 149 SLDVASGLGAPLEGVLVE 2123 150APLEGVLVELEVLPAAIP 5144 151 LEVLPAAIPLEAQNFSVP 3152 152LEAQNFSVPEGGSLTLAP 3277 153 EGGSLTLAPPLLRVSGPY 4455 154PLLRVSGPYFPTLLGLSL 4311 155 FPTLLGLSLQVLEPPQHG 3545 156QVLEPPQHGPLQKEDGPQ 1883 157 PLQKEDGPQARTLSAFSW 3132 158ARTLSAFSWRMVEEQLIR 3149 159 RMVEEQLIRYVHDGSETL 947 160YVHDGSETLTDSFVLMAN 1332 161 TDSFVLMANASEMDRQSH 320 162ASEMDRQSHPVAFTVTVL 521 163 PVAFTVTVLPVNDQPPIL 884 164 PVNDQPPILTTNTGLQMW867 165 TTNTGLQMWEGATAPIPA 1235 166 EGATAPIPAEALRSTDGD 1323 167EALRSTDGDSGSEDLVYT 1970 168 SGSEDLVYTIEQPSNGRV 1972 169IEQPSNGRVVLRGAPGTE 2836 170 VLRGAPGTEVRSFTQAQL 11671 171VRSFTQAQLDGGLVLFSH 2167 172 DGGLVLFSHRGTLDGGFP 2307 173RGTLDGGFPFRLSDGEHT 2979 174 FRLSDGSHTSPGHFFRVT 3900 175SPGHFFRVTAQKQVLLSL 4176 176 AQKQVLLSLKGSQTLTVC 3627 177KGSQTLTVCPGSVQPLSS 6489 178 PGSVQPLSSQTLRASSSA 3448 179QTLRASSSAGTDPQLLLY 1159 180 GTDPQLLLYRVVRGPQLG 1266 181RVVRGPQLGRLFHAQQDS 3735 182 RLFHAQQDSTGEALVNFT 1155 183TGEALVNFTQAEVYAGNI 1544 184 QAEVYAGNILYEHENPPE 889 185LYEHEMPPEPFWEAHDTL 826 186 PFWEAHDTLELQLSSPFA 1748 187ELQLSSPPARDVAATLAV 1713 188 RDVAATLAVAVSFEAACP 1953 189AVSFEAACPQRPSHLWKN 2533 190 QRPSHLWKNKGLWVPEGQ 5178 191KGLWVPEGQRARITVAAL 3891 192 RARITVAALDASNLLASV 5276 193DASNLLASVPSPQRSEHD 2460 194 PSPQRSEHDVLFQVTQFP 2205 195VLFQVTQFPSRGQLLVSE 2556 196 SRGQLLVSEEPLHAGQPH 1359 197EPLHAGQPHFLQSQLAAG 1265 198 FLQSQLAAGQLVYAHGGG 1361 199QLVYAHGGGGTQQDGFHF 1210 200 GTQQDGFHFRAHLQGPAG 3436 201RAHLQGPAGASVAGPQTS 3587 202 ASVAGPQTSEAFAITVRD 980 203EAFAITVRDVNERPPQPQ 1032 204 VNERPPQPQASVPLRLTR 4790 205ASVPLRLTRGSRAPISRA 4393 206 GSRAPISRAQLSVVDPDS 2547 207QLSVVDPDSAPGEIEYEV 1318 208 APGEIEYEVQRAPHNGFL 1561 209QRAPHNGFLSLVGGGLGP 4879 210 SLVGGGLGPVTRFTQADV 3371 211VTRFTQADVDSGRLAFVA 2747 212 DSGRLAFVANGSSVAGIF 5532 213NGSSVAGIFQLSMSDGAS 3503 214 QLSMSDGASPPLPMSLAV 2245 215PPLPMSLAVDILPSAIEV 1845 216 DILPSAIEVQLRAPLEVP 1504 217QLRAPLEVPQALGRSSLS 5177 218 QALGRSSLSQQQLRVVSD 3060 219QQQLRVVSDREEPEAAYR 988 220 REEPEAAYRLIQGPQYGH 762 221 LIQGPQYGHLLVGGRPTS1334 222 LLVGGRPTSAFSQFQIDQ 2308 223 AFSQFQIDQGEVVFAFTN 2915 224GEVVFAFTNFSSSHDHFR 3745 225 FSSSHDHFRVLALARGVN 2196 226VLALARGVNASAVVNVTV 1991 227 ASAVVNVTVRALLHVWAG 1402 228RALLHVWAGGPWPQGATL 1790 229 GPWPQGATLRLDFTVLDA 1447 230RLDPTVLDAGELANRTGS 1796 231 GELANRTGSVPRFRLLEG 7317 232VPRFRLLEGPRHGRVVRV 3761 233 PRHGRVVRVPRARTEPGG 8844 234PRARTEPGGSQLVEQFTQ 3609 235 SQLVEQFTQQDLEDGRLG 1985 236QDLEDGRLGLEVGRPEGR 1551 237 LEVGRPEGRAPGPAGDSL 1136 238APGPAGDSLTLELWAQGV 993 239 TLELWAQGVFPAVASLDF 844 240 PPAVASLDFATEPYNAAR1339 241 ATEPYNAARPYSVALLSV 786 242 PYSVALLSVPEAARTEAG 1723 243PEAARTEAGKPESSTPTG 1417 244 KPSSSTPTGEPGPMASSP 1449 245EPGPMASSPEPAVAKGGF 1739 246 EPAVAKGGFLSFLEANMF 4457 247LSFLEANMFSVIIPMCLV 1275 248 SVIIPMCLVLLLLALILP 1306 249LLLLALILPLLFYLRKRN 1291 250 LLFYLRKRNKTGKHDVQV 1820 251KTGKHDVQVLTAKPRNGL 13573 252 LTAKPRNGLAGDTETFRK 10322 253AGDTETFRKVEPGQAIPL 4744 254 VEPGQAIPLTAVPGQGPP 3571 255TAVPGQGPPPGGQPDPEL 1733 256 PGGQPDPELLQFCRTPNP 11325 257LQFCRTPNPALKNGQYWV 1550

FIG. 5 represents a graphical image of the binding data. 11BD-2E11-2bound most strongly to peptides #26, SEQ ID NO: 1 and #71, SEQ ID NO: 2.Weaker binding, which was greater than background, was recognizable onpeptides #3, SEQ ID NO: 3, #66, SEQ ID NO: 4, #170, SEQ ID NO: 5, #251,SEQ ID NO: 6, #252, SEQ ID NO: 7 and #256, SEQ ID NO: 8. These resultsindicated that 11BD-2E11-2 may bind to a discontinuous epitope with twomajor binding sites (peptides #26 and #71) as well as to a number ofother sites.

EXAMPLE 5

As outlined in Ser. No. 10/743,451, the hybridoma cell line 11BD-2E11-2was deposited, in accordance with the Budapest Treaty, with the AmericanType Culture Collection, University Blvd., Manassas, Va. 20110-2209 onNov. 11, 2003, under Accession Number PTA-5643. In accordance with CFR1.808, the depositors assure that all restrictions imposed on theavailability to the public of the deposited materials will beirrevocably removed upon the granting of a patent.

Antibody Production

11BD-2E11-2 monoclonal antibody was produced by culturing the hybridoma(PTA-5643) in CL-1000 flasks (BD Biosciences, Oakville, ON) withcollections and reseeding occurring twice/week. The antibody waspurified according to standard antibody purification procedures withProtein G Sepharose 4 Fast Flow (Amersham Biosciences, Baie d'Urfé, QC).

As previously described in Ser. No. 10/348,231, 11BD-2E11-2 was comparedto a number of both positive (anti-Fas (EOS9.1, IgM, kappa, 20micrograms/mL, eBioscience, San Diego, Calif.), anti-Her2/neu (IgG1,kappa, 10 microgram/mL, Inter Medico, Markham, ON), anti-EGFR(C225,IgG1, kappa, 5 microgram/mL, Cedarlane, Homby, ON), Cycloheximide (100micromolar, Sigma, Oakville, ON), NaN₃ (0.1%, Sigma, Oakville, ON)) andnegative (107.3 (anti-TNP, IgG1, kappa, 20 microgram/mL, BD Biosciences,Oakville, ON), G155-178 (anti-TNP, IgG2a, kappa, 20 microgram/mL, BDBiosciences, Oakville, ON), MPC-11 (antigenic specificity unknown,IgG2b, kappa, 20 microgram/mL), J606 (anti-fructosan, IgG3, kappa, 20microgram/mL), IgG Buffer (2%)) controls in a cytotoxicity assay (Table2). Breast cancer (MDA-MB-231 (MB-231), MDA-MB-468 (MB-468), MCF-7),colon cancer (HT-29, SW1116, SW620), lung cancer (NCI H460), ovariancancer (OVCAR-3 (OVCAR)), prostate cancer (PC-3), and non-cancer (CCD27sk, Hs888 Lu) cell lines were tested (all from the ATCC, Manassas,Va.). The Live/Dead cytotoxicity assay was obtained from MolecularProbes (Eugene, Oreg.). The assays were performed according to themanufacturer's instructions with the changes outlined below. Cells wereplated before the assay at the predetermined appropriate density. After2 days, purified antibody or controls were diluted into media, and then100 microliters were transferred to the cell plates and incubated in a 5percent CO₂ incubator for 5 days. The plate was then emptied byinverting and blotted dry. Room temperature DPBS containing MgCl₂ andCaCl₂ was dispensed into each well from a multi-channel squeeze bottle,tapped three times, emptied by inversion and then blotted dry. 50microliters of the fluorescent calcein dye diluted in DPBS containingMgCl₂ and CaCl₂ was added to each well and incubated at 37° C. in a 5percent CO₂ incubator for 30 minutes. The plates were read in aPerkin-Elmer HTS7000 fluorescence plate reader and the data was analyzedin Microsoft Excel and the results were tabulated in Table 3. The datarepresented an average of four experiments tested in triplicate andpresented qualitatively in the following fashion: 4/4 experimentsgreater than threshold cytotoxicity (+++), 3/4 experiments greater thanthreshold cytotoxicity (++), 2/4 experiments greater than thresholdcytotoxicity (+). Unmarked cells in Table 3 represent inconsistent oreffects less than the threshold cytotoxicity. 11BD-2E11-2 wasspecifically cytotoxic in breast and ovarian cancer cells, and did notaffect normal cells. The chemical cytotoxic agents induced theirexpected cytotoxicity while a number of other antibodies which wereincluded for comparison also performed as expected given the limitationsof biological cell assays. In toto, it was shown that the 11BD-2E11-2antibody has cytotoxic activity against two cancer cell types. Theantibody was selective in its activity since not all cancer cell typeswere susceptible. Furthermore, the antibody demonstrated functionalspecificity since it did not produce cytotoxicity against non-cancercell types, which is an important factor in a therapeutic situation.

TABLE 3 BREAST COLON LUNG OVARY PROSTATE NORMAL MB-231 MB-468 MCF-7HT-29 SW1116 SW620 NCI H460 OVCAR PC-3 CCD 27sk Hs888 Lu 11BD-2E11-2 −− + − − − − + − − − anti-Fas − − +++ − − − − +++ + − + anti-Her2 + − + −− − − + − − − anti-EGFR − +++ + − +++ − − + − + − CHX (100 μM) +++ ++++++ +++ +++ +++ +++ +++ +++ +++ +++ NaN₃ (0.1%) +++ +++ +++ +++ − − ++++++ +++ − − IgG1 +++ + IgG2a +++ + IgG2b +++ IgG3 IgG Buffer +

As previously described in Ser. No. 10/348,231 and Ser. No. 10/810,744,binding of 11BD-2E11-2 to the above-mentioned panel of cancer and normalcell lines plus the following additional ovarian cancer cell lines(A2780-cp, A2780-s, C-14, OV2008, Hey, OCC-1, OVCA-429 and ES-2+SEAP)was assessed by flow cytometry (FACS). Cells were prepared for FACS byinitially washing the cell monolayer with DPBS (without Ca⁺⁺ and Mg⁺⁺).Cell dissociation buffer (INVITROGEN, Burlington, ON) was then used todislodge the cells from their cell culture plates at 37° C. Aftercentrifugation and collection the cells were resuspended in Dulbecco'sphosphate buffered saline containing MgCl₂, CaCl₂ and 2 or 25 percentfetal bovine serum (FBS) at 4° C. (wash media) and counted, aliquoted toappropriate cell density, spun down to pellet the cells and resuspendedin staining media (DPBS containing MgCl₂ and CaCl₂+/−2 percent FBS)containing 11BD-2E11-2 or control antibodies (isotype control oranti-EGFR) at 20 μg/mL on ice for 30 min. Prior to the addition of AlexaFluor 488-conjugated secondary antibody the cells were washed once withwash media. The Alexa Fluor 488-conjugated antibody in staining mediawas then added for 20 to 30 min. The cells were then washed for thefinal time and resuspended in staining media containing 1 μg/mLpropidium iodide or 1.5 percent paraformaldehyde. Flow cytometricacquisition of the cells was assessed by running samples on a FACScanusing the CellQuest software (BD Biosciences, Oakville, ON). The forward(FSC) and side scatter (SSC) of the cells were set by adjusting thevoltage and amplitude gains on the FSC and SSC detectors. The detectorsfor the three fluorescence channels (FL1, FL2, and FL3) were adjusted byrunning cells stained with purified isotype control antibody followed byAlexa Fluor 488-conjugated secondary antibody such that cells had auniform peak with a median fluorescent intensity of approximately 1-5units. Live cells were acquired by gating for FSC and propidium iodideexclusion (when used). For each sample, approximately 10,000 live cellswere acquired for analysis and the resulted are presented in Tables 4and 5. Tables 4 and 5 tabulated the mean fluorescence intensity foldincrease above isotype control and is presented qualitatively as: lessthan 5 (−); 5 to 50 (+); 50 to 100 (++); above 100 (+++) and inparenthesis, the percentage of cells stained.

TABLE 4 BREAST LUNG NORMAL MB- COLON NCI OVARY PROSTATE CCD Hs888Antibody Isotype 231 MB-468 MCF-7 HT-29 SW1116 SW620 H460 OVCAR PC-327sk CCD-112 Lu 11BD-2E11-2 IgG1, k + − − − − − − − − + + + anti-EGFRIgG1, k ++ ++ − + + − + + + + + +

TABLE 5 Ovarian Antibody Isotype A2780-cp A2780-s C-14 OV2008 ES-2 +SEAP Hey OCC-1 OVCA-429 11BD-2E11-2 IgG1, k + + − − + + + − anti-EGFRIgG1, k − − + + + + + +

Representative histograms of 11BD-2E11-2 antibodies were compiled forFIG. 6. 11BD-2E11-2 displayed specific tumor binding to the breast tumorcell line MDA-MB-231 (Table 4) and several ovarian tumor cell linesincluding ES-2+SEAP (Table 5). There was also binding of 11BD-2E11-2 tonon-cancer cells, however that binding did not produce cytotoxicity.This was further evidence that binding was not necessarily predictive ofthe outcome of antibody ligation of its cognate antigen, and was anon-obvious finding. This suggested that the context of antibodyligation in different cells was determinative of cytoxicity rather thanjust antibody binding.

EXAMPLE 6 Normal Human Tissue Staining

IHC studies were conducted to characterize 11BD-2E11-2 antigendistribution in humans. IHC optimization studies were performedpreviously in order to determine the conditions for further experiments.11BD-2E11-2 monoclonal antibody was produced and purified as statedabove.

As disclosed in Ser. No. 10/810,744, binding of antibodies to 20 normalhuman tissues was performed using a frozen human normal organ tissuearray (Clinomics, Watervliet, N.Y.). Slides were postfixed for 10 min incold (−20° C.) acetone and then allowed to come to room temperature.Slides were rinsed in 4° C. cold phosphate buffered saline (PBS) 3 timesfor 2 min each followed by blocking endogenous peroxidase activity withwashing in 3 percent hydrogen peroxide for 10 min. Slides were thenrinsed in PBS 3 times for 5 min followed by incubation in Universalblocking solution (Dako, Toronto, Ontario) for 5 min at roomtemperature. 11BD-2E11-2, anti-human muscle actin (Clone HHF35, Dako,Toronto, Ontario) or isotype control antibody (directed towardsAspergillus niger glucose oxidase, an enzyme which is neither presentnor inducible in mammalian tissues; Dako, Toronto, Ontario) were dilutedin antibody dilution buffer (Dako, Toronto, Ontario) to its workingconcentration (5 μg/mL for each antibody except for anti-actin which was2 μg/mL) and incubated overnight for 1 hr at room temperature. Theslides were washed with PBS 3 times for 2 minutes each. Immunoreactivityof the primary antibodies was detected/visualized with HRP conjugatedsecondary antibodies as supplied (Dako Envision System, Toronto,Ontario) for 30 min at room temperature. Following this step the slideswere washed with PBS 3 times for 2 min each and a color reactiondeveloped by adding DAB (3,3′-diaminobenzidine tetrahydrachloride, Dako,Toronto, Ontario) chromogen substrate solution for immunoperoxidasestaining for 10 min at room temperature. Washing the slides in tap waterterminated the chromogenic reaction. Following counterstaining withMeyer's Hematoxylin (Sigma Diagnostics, Oakville, ON), the slides weredehyrdated with graded ethanols (95-100%) and cleared with xylene. Usingmounting media (Dako Faramount, Toronto, Ontario) the slides werecoverslipped. Slides were microscopically examined using an Axiovert 200(Zeiss Canada, Toronto, ON) and digital images acquired and stored usingNorthern Eclipse Imaging Software (Mississauga, ON). Results were read,scored and interpreted by a pathologist.

Table 6 presents a summary of the results of 11BD-2E11-2 staining of anarray of normal human tissues. From the table, there were 2 maincategories of tissue staining. A group of tissues was completelynegative. These tissues included normal thyroid, bronchus and cardiacmuscle of the left ventricle (FIG. 7). The second group of tissuesincluded tissues in which staining was positive in the tissue section,but was limited to smooth muscle fibers of blood vessels and/or theepithelium (FIG. 8). These results suggested that the antigen for11BD-2E11-2 was not widely expressed on normal tissues, and that theantibody would bind only to a limited number of tissues in humans. Thenormal human tissue staining of 11BD-2E11-2 resembles that previouslyreported for an anti-MCSP antibody; B5. B5 was previously shown to bindto skin keratinocytes, lung alveolar epithelium and capillaryendothelium.

TABLE 6 11BD-2E11-2 IHC on Frozen Human Normal Tissue IHC Score Datasheet IgG negative S. No. Tissues Age Sex 11BD-2E11-2 Actin control 1Bronchus 61 M − (PD) +++ SMF &Myoepithelium of CD mucus acini 2Diaphragm 61 M +++ SMF of blood vessels +/− Skeletal muscle fibers +++Skeletal muscle fibers & − SMF of blood vessels 3 Pectoral muscle 61 M+++ SMF of blood vessels +++ Skeletal muscle fibers & − (Skeletalmuscle) SMF of blood vessels 4 Lung 61 M +++ Alveolar epithelium &SMF ofblood vessels CD − (F) 5 Aorta 61 M ++ SMF (F) CD − 6 Left ventricle 61M − +++ SMF of blood vessels − (Cardiac muscle) + Cardiac muscle fibers7 Esophagus 61 M +++ SMF (PD) CD − (F) 8 Trachea 61 M − (PD) +++ SMF&myoepithelium of − mucus acini 9 Kidney 61 M +++ SMF of blood vessels+++ SMF of blood vessels − 10 Adrenal 61 M +++ SMF of blood vessels +++SMF of blood vessels − 11 Pancreas 61 M +++ SMF of blood vessels +Acinar epithelium +++ SMF of blood vessels − 12 Spleen 61 M +++ SMF ofblood vessels &Polymorphs (F) +++ SMF of blood vessels, Bg (polymorphs)reticular fibers &polymorphs (F) 13 Liver 61 M +++ SMF of blood vessels− (PD) − 14 Skin 61 M +++ SMF of blood vessels +/− Keratinocytes +++ SMFof blood vessels Bg (Stroma) 15 Colon 61 M +++ SMF of blood vessels +++SMF − 16 Thyroid 61 M − (PD) − (PD) − 17 Prostate 61 M ++ SMF of bloodvessels +/− Glandular epithelium CD CD 18 Testicle 61 M ++ SMF of bloodvessels +++ stromal cells − 19 Breast 61 M +/− Ductal epithelium +++ SMFof blood vessels − +++ SMF of blood vessels 20 Ovary 80 F ++ SMF ofblood vessels &Stroma F CD Abbreviations: SMF: smooth muscle fiber, Bg:background staining, PD: partially detached F: folded, CD: completelydetached.

EXAMPLE 7 Human Breast Tumor Tissue Staining

An IHC study was undertaken to determine the cancer association of the11BD-2E11-2 antigen with human breast cancers (disclosed in Ser. No.10/810,744). A comparison was made for actin (positive control), and anantibody directed towards Aspergillus niger glucose oxidase, an enzymewhich is neither present nor inducible in mammalian tissues (negativecontrol). A breast cancer tissue array derived from 15 breast cancerpatients and 5 samples derived from non-neoplastic breast tissue inbreast cancer patients were used (Clinomics, Watervliet, N.Y.). Thefollowing information was provided for each patient: age, sex, anddiagnosis. The procedure for IHC from Example 6 was followed.

Table 7 provides a binding summary of 11BD-2E11-2 antibody staining of abreast cancer tissue array. Each array contained tumor samples from 15individual patients. Overall, 62 percent of the 8 (7 of the tissuesamples were either completely detached or not representative) patientstested were positive for the 11BD-2E11-2 antigen. Also for 11BD-2E11-2,0 out of 3 (again 2 of the tissue samples were completely detached)normal breast tissue samples from breast cancer patients were positive(FIG. 9). For the 11BD-2E11-2 antigen there did not appear to be a trendto greater positive expression with higher tumor stage. However, thisresult was limited due to the small sample size. The 11BD-2E11-2staining was specific for cancerous cells (FIG. 9). The stainingpattern, from 11BD-2E11-2, showed that in patient samples, the antibodywas highly specific for malignant cells thereby making it an attractivedruggable target. The breast tumor tissue staining of 11BD-2E11-2resembles that previously reported for the anti-MCSP antibody B5. B5 waspreviously shown to bind to 60 percent of breast carcinoma tumor tissue.

TABLE 7 11BD-2E11-2 IHC on Frozen Human Normal and Breast Tumor TissueData Sheet IHC Score S. NO. Tissue Age Sex Diagnosis 11BD-2E11-2 ActinIgG negative control 1 Breast 61 F Infiltrating Ductal Carcinoma CD CDCD 2 Breast 74 F Infiltrating Ductal Carcinoma − (PD) − Tumor +++ SMF ofblood − vessels 3 Breast 60 F Infiltrating Ductal Carcinoma CD PD CD 4Breast 69 F Infiltrating Ductal Carcinoma NR NR − 5 Breast 64 FInfiltrating Ductal Carcinoma CD − CD 6 Breast 65 F Medullary Carcinoma+++ (Tumor cells) − − 7 Breast 75 F Infiltrating Ductal Carcinoma +++(Tumor cells) CD − 8 Breast 48 F Infiltrating Ductal Carcinoma ++ (Tumorcells) − Tumor ++ Stroma − 9 Breast 87 F Infiltrating Ductal Carcinoma+/− (Tumor cells) − Tumor +++− SMF of blood CD vessels 10 Breast 75 FInfiltrating Ductal Carcinoma NR (+/− SMF of CD − blood vessels) 11Breast 76 F Infiltrating Ductal Carcinoma − − Tumor +++ SMF of blood −vessels &stroma 12 Breast 66 F Infiltrating Ductal Carcinoma CD CD − 13Breast 58 F Infiltrating Ductal Carcinoma +++ (Tumor cells) CD CD 14Breast 37 F Infiltrating Ductal Carcinoma CD − Tumor +++ Stroma − 15Breast 70 F Infiltrating Ductal Carcinoma − − Tumor +++ Myoepithelium CD&SMF of blood vessels 16 Breast 48 F Normal − (PD) CD CD 17 Breast 60 FNormal − − (PD) − 18 Breast 30 F Normal CD − Tumor +++ Myoepithelium&SMF of blood vessels 19 Breast 34 F Normal CD − Tumor ++ Myoepithelium(PD) 20 Breast 43 F Normal − − Tumor + SMF of blood vesselsAbbreviations: SMF: smooth muscle fiber, PD: partially detached, F:folded, CD: completely detached.

EXAMPLE 8

An IHC study was undertaken to determine the cancer association of the11BD-2E11-2 antigen with human melanoma cancers. A comparison was madefor an anti-CD63 antibody (NIK-C3; MEDICORP, Montreal QC); positivecontrol), and an antibody directed towards Aspergillus niger glucoseoxidase, an enzyme which is neither present nor inducible in mammaliantissues (negative control). A melanoma cancer tissue array derived from35 melanoma cancer patients and 10 samples derived from normal skintissue in melanoma cancer patients was used (TriStar Technology Group,LLC, Bethesda, Md.). The procedure for IHC from Example 6 was followedexcept for the following modifications. The color reaction developed byadding AEC (Dako, Toronto, Ontario) chromogen substrate solution forimmunoperoxidase staining for 10 minutes at room temperature. Washingthe slides in tap water terminated the chromogenic reaction. Followingcounterstaining with Meyer's Hematoxylin (Sigma Diagnostics, Oakville,ON), the slides were cleared with distilled water.

Table 8 provides a binding summary of 11BD-2E11-2 antibody staining of amelanoma cancer tissue array. Each array contained tumor samples from 35individual patients and normal skin from 10 patients. Overall, 67percent of the 33 (2 of the tissue samples were completely pigmented)patients tested were positive for the 11BD-2E11-2 antigen (FIG. 10). Inaddition, 0 out of 6 (4 of the tissue samples were non representative ornot available) normal skin tissue samples from melanoma cancer patientswere positive (FIG. 11). The 11BD-2E11-2 staining was specific forcancerous cells (FIG. 11). The staining pattern, from 11BD-2E11-2,showed that in patient samples, the antibody was highly specific formalignant cells thereby making it an attractive druggable target anddemonstrating the utility of 11BD-2E11-2 as a potential drug.

TABLE 8 11BD-2E11-2 IHC on Frozen Human Normal Skin and Melanoma TumorTissue IHC observations IgG negative Coordinates Primary/meta Organ11B-2E11-2 NKI-C3 control A1a meta lymph node Completely CompletelyCompletely Pigmented Pigmented Pigmented A1b meta lymph node − + − A1cmeta spleen +++ +++ − A1d primary skin + ++ − A1e primary esophagus +/−+++ − A1f meta lymph node +++ ++ − A1g primary skin + +++ − A1h metalung ++ +++ − A1i meta lymph node +/− − cd A1k meta lymph node ++ ++ −A2a primary skin Completely Completely Completely Pigmented PigmentedPigmented A2b meta skin − − − A2c primary skin +++ − − A2d meta softtissue +++ ++ − A2e meta lymph node +++ +++ − A2f meta lymph node ++++++ cd A2g primary skin − +++ − A2h meta lymph node + − − A2i meta lymphnode +++ + − A2k meta soft tissue − − cd A3a primary skin +++ ++ − A3bprimary skin +++ +++ − A3c meta lymph node − − − A3d meta lymph node ++/− − A3e meta lymph node +/− +++ − A3f meta lymph node +/− ++ − A3gmeta lymph node − − − A3h meta lymph node − − − A3i meta lymph node − −− A3k meta lymph node − − − A4a meta lymph node − +/− − A4b meta lymphnode +++ +++ − A4c primary skin + +++ − A4d meta soft tissue +++ +++ −A4e meta lymph node − ++ − A5a normal skin − − − A5b normal skin − − −A5c normal skin NR NR NR A5d normal skin − − − A5e normal skin − − − A5fnormal skin − − − A5g normal skin NA NA NA A5h normal skin − − − A5inormal skin NR NR NR A5k normal skin NR NR cd Abbreviations: meta:metastatic, NR: section is not representative, cd: section is completelydetached, NA: section is not available.

EXAMPLE 9 In Vivo MDA-MB-468 Established Tumor Experiment

As disclosed in Ser. No. 10/810,744 and with reference to FIG. 12, 6 to8 week old female SCID mice were implanted with 2 million MDA-MB-468human breast cancer cells in 100 microlitres saline injectedsubcutaneously in the scruff of the neck. Tumor growth was measured withcalipers every week. When the majority of the cohort reached a tumorvolume of 100 mm³, 5-6 mice were randomized into each of 2 treatmentgroups. 11BD-2E11-2 or buffer control was administered intraperitoneallywith 10 mg/kg/dose at a volume of 300 microliters after dilution fromthe stock concentration with a diluent that contained 2.7 mM KCl, 1 mMKH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄. The antibodies were thenadministered 3 times per week for a total of 10 doses in the samefashion until day 66 post-implantation. Tumor growth was measured aboutevery seventh day with calipers for the duration of the study or untilindividual animals reached CCAC end-points. Body weights of the animalswere recorded for the duration of the study. At the end of the study allanimals were euthanised according to CCAC guidelines.

At the time of randomization the mean tumor volumes and the standarddeviations in each group were similar. Statistically there was nodifference in body weight between the groups. This indicated that truerandomization had occurred. As shown in FIG. 12, the antibody11BD-2E11-2 suppressed tumor growth by 25 percent in comparison tobuffer control at the end of the 3-week treatment period (p=0.52).Although this was not a significant difference, a trend towards reducedtumor volume in comparison to the buffer control was observed throughoutthe study. Therefore, II BD-2E11-2 has shown efficacy in an establishedbreast cancer model.

EXAMPLE 10 In Vivo ES-2+SEAP Established Tumor Experiment

As disclosed in Ser. No. 10/810,744 and with reference to FIGS. 13 and14, 6 to 8 week old female athymic nude mice were intraperitoneallyimplanted with 10 million ES-2+SEAP human ovarian cancer cells stablytransfected to express human placental secreted alkaline phosphatase(SEAP). The 10 million ovarian cancer cells were resuspended in 500microlitres serum-free α-MEM. Tumor growth was confirmed with thesacrifice of 3 mice on day 7. Following the confirmation of tumor growthon day 7, 8 mice were randomized into each of 2 treatment groups.11BD-2E11-2 or buffer control was administered intraperitoneally with 10mg/kg/dose at a volume of 250 microliters after dilution from the stockconcentration with a diluent that contained 2.7 mM KCl, 1 mM KH₂PO₄, 137mM NaCl and 20 mM Na₂HPO₄. The antibodies were then administered onceper day for 5 doses and then once every other day for another 5 dosesfor a total of 10 doses. Tumor burden was extrapolated by measuringcirculating SEAP levels and assessed visually upon necropsy for theduration of the study or until individual animals reached CCACend-points. Body weights of the animals were recorded for the durationof the study. At the end of the study all animals were euthanisedaccording to CCAC guidelines.

At the time of randomization circulating plasma SEAP levels (indicativeof tumor burden) were analyzed. There was not a significant differencein the average SEAP level between the 11BD-2E11-2 and buffer controltreatment group. However, within groups there was variable tumortake-rate. As shown in FIG. 13, the antibody 11BD-2E11-2 displayed atrend for improved survival in a cohort of the treatment group. Asillustrated in FIG. 14, one animal receiving 11BD-2E11-2 treatment had adecreased amount of circulating SEAP to nearly negligible levels. Thelow level of circulating SEAP continued on until approximately 60 dayspost-implantation.

EXAMPLE 11 In Vivo A2058 Human Melanoma Preventative Tumor Experiment

With reference to the data shown in FIG. 15, 4 to 8 week old, femaleSCID mice were implanted with 0.75 million A2058 human melanoma cancercells in 100 microliters saline injected subcutaneously in the scruff ofthe neck. The mice were randomly divided into 2 treatment groups of 5.On the day after implantation 20 mg/kg of 111BD-2E11-2 test antibody orbuffer control was administered intraperitoneally at a volume of 300microliters after dilution from the stock concentration with a diluentthat contained 2.7 mM KCl, 1 mM KH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄.The antibody or buffer control was then administered once per week for aperiod of 7 weeks in the same fashion.

Tumor growth was measured about every 7th day with calipers for up to 10weeks or until individual animals reached the Canadian Council forAnimal Care (CCAC) end-points. Body weights of the animals were recordedfor the duration of the study. At the end of the study all animals wereeuthanised according to CCAC guidelines.

As shown in FIG. 15, 11BD-2E11-2 treatment resulted in decreased tumorgrowth compared to treatment with the buffer control. On day 55 (5 daysafter the end of treatment), the mean tumor volume in the 11BD-2E11-2treated group was 58 percent of the buffer control (p=0.046, unpairedt-test). Therefore, 11BD-2E11-2 displayed efficacy in the treatment ofbreast, ovarian and melanoma in vivo models of human cancer and reducedtumor burdens in comparison to controls in those same cancers.

EXAMPLE 12 In Vivo A2058 Human Melanoma Established Tumor Experiment

With reference to FIG. 16, 6 to 8 week old female SCID mice wereimplanted with 0.5 million A2058 human melanoma cancer cells in 100microlitres saline injected subcutaneously in the scruff of the neck.Tumor growth was measured with calipers every week. When the majority ofthe cohort reached a tumor volume of 100 mm³, 5 mice were randomizedinto each of 2 treatment groups. 11BD-2E11-2 or buffer control wasadministered intraperitoneally with 20 mg/kg/dose at a volume of 300microliters after dilution from the stock concentration with a diluentthat contained 2.7 mM KCl, 1 mM KH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄.The antibodies were then administered 3 times per week for a total of 10doses in the same fashion until day 44 post-implantation. Tumor growthwas measured about every seventh day with calipers for the duration ofthe study or until individual animals reached CCAC end-points. Bodyweights of the animals were recorded for the duration of the study. Atthe end of the study all animals were euthanised according to CCACguidelines.

At the time of randomization the mean tumor volumes and the standarddeviations in each group were similar. Statistically there was nodifference in body weight between the groups. This indicated that truerandomization had occurred. As shown in FIG. 13, the antibody11BD-2E11-2 suppressed tumor growth by 49 percent in comparison tobuffer control after the treatment period (p=0.1272; unpaired t-test).Although this was not a significant difference, a trend towards reducedtumor volume in comparison to the buffer control was observed throughoutthe study. Therefore, 11BD-2E11-2 has shown efficacy in both anestablished breast, ovarian and melanoma cancer model. In all, theseresults in which 11BD-2E11-2 produced benefits (improved survival and/ordecreased tumor burden in comparison to control treatment) in multiplemodels of human cancer suggest pharmacologic and pharmaceutical benefitsof this antibody for cancer therapy in mammals, including man.

The preponderance of evidence shows that 11BD-2E11-2 mediatesanti-cancer effects through ligation of an epitope present on MCSP. Forthe purpose of this invention, said epitope is defined as a “MCSPantigenic moiety” characterized by its ability to bind with a monoclonalantibody encoded by the hybridoma cell line 11BD-2E11-2, antigenicbinding fragments thereof or antibody conjugates thereof. It has beenshown, in Example 3, 11BD-2E11-2 antibody can be used toimmunoprecipitate the cognate antigen from expressing cells such asMDA-MB-231 cells. Further it could be shown that the 11BD-2E11-2antibody could be used in detection of cells and/or tissues whichexpress a MCSP antigenic moiety which specifically binds thereto,utilizing techniques illustrated by, but not limited to FACS, cell ELISAor IHC.

Thus, it could be shown that the immunoprecipitated 11BD-2E11-2 antigencan inhibit the binding of 11BD-2E11-2 to such cells or tissues usingFACS, cell ELISA or IHC assays. Further, as with the 11BD-2E11-2antibody, other anti-MCSP antibodies could be used to immunoprecipitateand isolate other forms of the MCSP antigen, and the antigen can also beused to inhibit the binding of those antibodies to the cells or tissuesthat express the antigen using the same types of assays.

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that while a certain form of the invention isillustrated, it is not to be limited to the specific form or arrangementof parts herein described and shown. It will be apparent to thoseskilled in the art that various changes may be made without departingfrom the scope of the invention and the invention is not to beconsidered limited to what is shown and described in the specification.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Anyoligonucleotides, peptides, polypeptides, biologically relatedcompounds, methods, procedures and techniques described herein arepresently representative of the preferred embodiments, are intended tobe exemplary and are not intended as limitations on the scope. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the appended claims. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A method for treating a patient suffering from a cancerous diseasecomprising: administering to said patient an anti-cancer antibody orantigen binding fragment thereof produced in accordance with a methodfor the production of anti-cancer antibodies which are useful intreating a cancerous disease, said antibody or antigen binding fragmentthereof characterized as being cytotoxic against cells of a canceroustissue, and essentially benign to non-cancerous cells; wherein saidantibody or antigen binding fragment thereof is placed in admixture witha pharmaceutically acceptable adjuvant and is administered in an amounteffective to mediate treatment of said cancerous disease; said antibodybeing an isolated monoclonal antibody or antigen binding fragmentthereof which binds to an antigenic moiety expressed by said canceroustissue, said antigenic moiety characterized as being bound by anisolated monoclonal antibody or antigen binding fragment thereofproduced by the hybridoma deposited with the ATCC as PTA-5643.
 2. Themethod for treating a patient suffering from a cancerous disease inaccordance with claim 1, wherein said isolated monoclonal antibody orantigen binding fragment thereof is a humanized or chimeric antibody ofthe isolated monoclonal antibody produced by the hybridoma depositedwith the ATCC as PTA-5643 or an antigen binding fragment thereof.
 3. Themethod for treating a patient suffering from a cancerous disease inaccordance with claim 1 comprising: conjugating said antibody or antigenbinding fragment thereof with a member selected from the groupconsisting of toxins, enzymes, radioactive compounds, and hematogenouscells, thereby forming an antibody conjugate; and administering saidantibody conjugate or conjugated fragments to said patient; wherein saidantibody conjugate or conjugated fragments are placed in admixture witha pharmaceutically acceptable adjuvant and are administered in an amounteffective to mediate treatment of said cancerous disease.
 4. The methodof claim 3, wherein said isolated monoclonal antibody or antigen bindingfragment thereof is a humanized or chimeric antibody of the isolatedmonoclonal antibody produced by the hybridoma deposited with the ATCC asPTA-5643 or an antigen binding fragment thereof.
 5. The method fortreating a patient suffering from a cancerous disease in accordance withclaim 1 wherein: the cytotoxicity of said antibody or antigen bindingfragment thereof is mediated through antibody dependent cellulartoxicity.
 6. The method for treating a patient suffering from acancerous disease in accordance with claim 1 wherein: the cytotoxicityof said antibody or antigen binding fragment thereof is mediated throughcomplement dependent cellular toxicity.
 7. The method for treating apatient suffering from a cancerous disease in accordance with claim 1wherein: the cytotoxicity of said antibody or antigen binding fragmentthereof is mediated through catalyzing of the hydrolysis of cellularchemical bonds.
 8. The method for treating a patient suffering from acancerous disease in accordance with claim 1 wherein: the cytotoxicityof said antibody or antigen binding fragment thereof is mediated throughproducing an immune response against putative cancer antigens residingon tumor cells.
 9. The method for treating a patient suffering from acancerous disease in accordance with claim 1 wherein: the cytotoxicityof said antibody or antigen binding fragment thereof is mediated throughtargeting of cell membrane proteins to interfere with their function.10. The method for treating a patient suffering from a cancerous diseasein accordance with claim 1 wherein: the cytotoxicity of said antibody orantigen binding fragment thereof is mediated through production of aconformational change in a cellular protein effective to produce asignal to initiate cell-killing.
 11. The method for treating a patientsuffering from a cancerous disease in accordance with claim 1 wherein:said method of production utilizes a tissue sample containing cancerousand non-cancerous cells obtained from a particular individual.
 12. Amethod for treating a patient suffering from a cancerous diseasecomprising: administering to said patient an antibody or antigen bindingfragment thereof produced in accordance with a method for the productionof anti-cancer antibodies which are useful in treating a cancerousdisease, said antibody being cytotoxic against cells of a canceroustissue, and essentially benign to non-cancerous cells; wherein saidantibody is the isolated monoclonal antibody encoded by the clonedeposited with the ATCC as PTA-5643 or an antigen binding fragmentthereof, and is placed in admixture with a pharmaceutically acceptableadjuvant and is administered in an amount effective to mediate treatmentof said cancerous disease.
 13. The method for treating a patientsuffering from a cancerous disease in accordance with claim 12, whereinsaid isolated monoclonal antibody or antigen binding fragment thereof isa humanized or chimeric antibody of the isolated monoclonal antibodyproduced by the hybridoma deposited with the ATCC as PTA-5643 or anantigen binding fragment thereof.
 14. The method for treating a patientsuffering from a cancerous disease in accordance with claim 12comprising: conjugating said antibody or antigen binding fragmentthereof with a member selected from the group consisting of toxins,enzymes, radioactive compounds, and hematogenous cells, whereby anantibody conjugate is formed; and administering said antibody conjugatesor antigen binding fragments thereof to said patient; wherein saidconjugated antibodies are placed in admixture with a pharmaceuticallyacceptable adjuvant and are administered in an amount effective tomediate treatment of said cancerous disease.
 15. The method of claim 14,wherein said isolated monoclonal antibody or antigen binding fragmentthereof is a humanized or chimeric antibody of the isolated monoclonalantibody produced by the hybridoma deposited with the ATCC as PTA-5643or an antigen binding fragment thereof.
 16. The method for treating apatient suffering from a cancerous disease in accordance with claim 12wherein: the cytotoxicity of said antibody or antigen binding fragmentthereof is mediated through antibody dependent cellular toxicity. 17.The method for treating a patient suffering from a cancerous disease inaccordance with claim 12 wherein: the cytotoxicity of said antibody orantigen binding fragment thereof is mediated through complementdependent cellular toxicity.
 18. The method for treating a patientsuffering from a cancerous disease in accordance with claim 12 wherein:the cytotoxicity of said antibody or antigen binding fragment thereof ismediated through catalyzing of the hydrolysis of cellular chemicalbonds.
 19. The method for treating a patient suffering from a cancerousdisease in accordance with claim 12 wherein: the cytotoxicity of saidantibody or antigen binding fragment thereof is mediated throughproducing an immune response against putative cancer antigens residingon tumor cells.
 20. The method for treating a patient suffering from acancerous disease in accordance with claim 12 wherein: the cytotoxicityof said antibody or antigen binding fragment thereof is mediated throughtargeting of cell membrane proteins to interfere with their function.21. The method for treating a patient suffering from a cancerous diseasein accordance with claim 12 wherein: the cytotoxicity of said antibodyor antigen binding fragment thereof is mediated through production of aconformational change in a cellular protein effective to produce asignal to initiate cell-killing.
 22. The method for treating a patientsuffering from a cancerous disease in accordance with claim 12 wherein:said method of production utilizes a tissue sample containing cancerousand non-cancerous cells obtained from a particular individual.
 23. Aprocess for mediating cytotoxicity of a human tumor cell which expressesan MCSP antigenic moiety on the cell surface comprising: contacting saidtumor cell with an isolated monoclonal antibody or antigen bindingfragment thereof, said antibody or antigen binding fragment thereofbeing an isolated monoclonal antibody or antigen binding fragmentthereof which binds to said expressed MCSP antigenic moiety, saidantigenic moiety characterized as being bound by the isolated monoclonalantibody produced by the hybridoma deposited with the ATCC as PTA-5643,whereby cell cytotoxicity occurs as a result of said binding.
 24. Theprocess of claim 23 wherein said isolated monoclonal antibody or antigenbinding fragment thereof is a humanized or chimeric antibody of theisolated monoclonal antibody produced by the hybridoma deposited withthe ATCC as PTA-5643 or an antigen binding fragment thereof.
 25. Theprocess of claim 23 wherein said isolated antibody or antigen bindingfragments thereof are conjugated with a member selected from the groupconsisting of cytotoxic moieties, enzymes, radioactive compounds, andhematogenous cells, whereby an antibody conjugate is formed.
 26. Theprocess of claim 25 wherein said isolated monoclonal antibody or antigenbinding fragment thereof is a humanized or chimeric antibody of theisolated monoclonal antibody produced by the hybridoma deposited withthe ATCC as PTA-5643 or an antigen binding fragment thereof.
 27. Theprocess of claim 23 wherein said isolated antibody or antigen bindingfragments thereof are murine.
 28. The process of claim 23 wherein thehuman tumor tissue sample is obtained from a tumor originating in atissue selected from the group consisting of breast, ovarian or melanomatissue.
 29. A method of extending survival and/or delaying diseaseprogression by treating a human tumor in a mammal, wherein said tumorexpresses an antigen which specifically binds to an isolated monoclonalantibody or antigen binding fragment thereof produced by the hybridomadeposited with the ATCC as accession number PTA-5643 comprisingadministering to said mammal said monoclonal antibody in an amounteffective to reduce said mammal's tumor burden, whereby diseaseprogression is delayed and/or survival is extended.
 30. The method ofclaim 29 wherein said antibody is conjugated to a cytotoxic moiety. 31.The method of claim 30 wherein said cytotoxic moiety is a radioactiveisotope.
 32. The method of claim 29 wherein said antibody activatescomplement.
 33. The method of claim 29 wherein said antibody mediatesantibody dependent cellular cytotoxicity.
 34. The method of claim 29wherein said antibody is a murine antibody.
 35. The method of claim 29wherein said isolated monoclonal antibody or antigen binding fragmentthereof is a humanized antibody of the isolated monoclonal antibodyproduced by the hybridoma deposited with the ATCC as PTA-5643 or anantigen binding fragment thereof.
 36. The method of claim 29 whereinsaid isolated monoclonal antibody or antigen binding fragment thereof isa chimeric antibody of the isolated monoclonal antibody produced by thehybridoma deposited with the ATCC as PTA-5643 or an antigen bindingfragment thereof.